Diverse branching forms regulated by a core auxin transport mechanism in plants.

Diverse branching forms have evolved multiple times across the tree of life to facilitate resource acquisition and exchange with the environment. In the vascular plant group, the ancestral pattern of branching involves dichotomy of a parent shoot apex to form two new daughter apices. The molecular basis of axillary branching in Arabidopsis is well understood, but few regulators of dichotomous branching are known. Through analyses of dichotomous branching in the lycophyte, Selaginella kraussiana, we identify PIN-mediated auxin transport as an ancestral branch regulator of vascular plants. We show that short-range auxin transport out of the apices promotes dichotomy and that branch dominance is globally coordinated by long-range auxin transport. Uniquely in Selaginella, angle meristems initiate at each dichotomy, and these can develop into rhizophores or branching angle shoots. We show that long-range auxin transport and a transitory drop in PIN expression are involved in angle shoot development. We conclude that PIN-mediated auxin transport is an ancestral mechanism for vascular plant branching that was independently recruited into Selaginella angle shoot development and seed plant axillary branching during evolution.

The TOPLESS corepressor regulates developmental switches in the bryophyte Physcomitrium patens that were critical for plant terrestrialisation.

The plant-specific TOPLESS (TPL) family of transcriptional corepressors is integral to multiple angiosperm developmental processes. Despite this, we know little about TPL function in other plants. To address this gap, we investigated the roles TPL plays in the bryophyte Physcomitrium patens, which diverged from angiosperms approximately 0.5 billion years ago. Although complete loss of PpTPL function is lethal, transgenic lines with reduced PpTPL activity revealed that PpTPLs are essential for two fundamental developmental switches in this plant: the transitions from basal photosynthetic filaments (chloronemata) to specialised foraging filaments (caulonemata) and from two-dimensional (2D) to three-dimensional (3D) growth. Using a transcriptomics approach, we integrated PpTPL into the regulatory network governing 3D growth and we propose that PpTPLs represent another important class of regulators that are essential for the 2D-to-3D developmental switch. Transcriptomics also revealed a previously unknown role for PpTPL in the regulation of flavonoids. Intriguingly, 3D growth and the formation of caulonemata were crucial innovations that facilitated the colonisation of land by plants, a major transformative event in the history of life on Earth. We conclude that TPL, which existed before the land plants, was co-opted into new developmental pathways, enabling phytoterrestrialisation and the evolution of land plants.

How was apical growth regulated in the ancestral land plant? Insights from the development of non-seed plants.

Land plant life cycles are separated into distinct haploid gametophyte and diploid sporophyte stages. Indeterminate apical growth evolved independently in bryophyte (moss, liverwort, and hornwort) and fern gametophytes, and tracheophyte (vascular plant) sporophytes. The extent to which apical growth in tracheophytes co-opted conserved gametophytic gene networks, or exploited ancestral sporophytic networks, is a long-standing question in plant evolution. The recent phylogenetic confirmation of bryophytes and tracheophytes as sister groups has led to a reassessment of the nature of the ancestral land plant. Here, we review developmental genetic studies of apical regulators and speculate on their likely evolutionary history.

CLAVATA modulates auxin homeostasis and transport to regulate stem cell identity and plant shape in a moss.

The CLAVATA pathway is a key regulator of stem cell function in the multicellular shoot tips of Arabidopsis, where it acts via the WUSCHEL transcription factor to modulate hormone homeostasis. Broad-scale evolutionary comparisons have shown that CLAVATA is a conserved regulator of land plant stem cell function, but CLAVATA acts independently of WUSCHEL-like (WOX) proteins in bryophytes. The relationship between CLAVATA, hormone homeostasis and the evolution of land plant stem cell functions is unknown. Here we show that in the moss, Physcomitrella (Physcomitrium patens), CLAVATA affects stem cell activity by modulating hormone homeostasis. CLAVATA pathway genes are expressed in the tip cells of filamentous tissues, regulating cell identity, filament branching, plant spread and auxin synthesis. The receptor-like kinase PpRPK2 plays the major role, and Pprpk2 mutants have abnormal responses to cytokinin, auxin and auxin transport inhibition, and show reduced expression of PIN auxin transporters. We propose a model whereby PpRPK2 modulates auxin gradients in filaments to determine stem cell identity and overall plant form. Our data indicate that CLAVATA-mediated auxin homeostasis is a fundamental property of plant stem cell function, probably exhibited by the last shared common ancestor of land plants.

How to build an effective research network: lessons from two decades of the GARNet plant science community.

Successful collaborative research is dependent on excellent ideas and innovative experimental approaches, as well as the provision of appropriate support networks. Collaboration requires venues, infrastructures, training facilities, and, perhaps most importantly, a sustained commitment to work together as a community. These activities do not occur without significant effort, yet can be facilitated and overseen by the leadership of a research network that has a clearly defined role to help build resources for their community. Over the past 20 years, this is a role that the UKRI-BBSRC-funded GARNet network has played in the support of the UK curiosity-driven, discovery-led plant science research community. This article reviews the lessons learnt by GARNet in the hope that they can inform the practical implementation of current and future research networks.

Positional cues regulate dorsal organ formation in the liverwort Marchantia polymorpha.

Bryophytes and vascular plants represent the broadest evolutionary divergence in the land plant lineage, and comparative analyses of development spanning this divergence therefore offer opportunities to identify truisms of plant development in general. In vascular plants, organs are formed repetitively around meristems at the growing tips in response to positional cues. In contrast, leaf formation in mosses and leafy liverworts occurs from clonal groups of cells derived from a daughter cell of the apical stem cell known as merophytes, and cell lineage is a crucial factor in repetitive organ formation. However, it remains unclear whether merophyte lineages are a general feature of repetitive organ formation in bryophytes as patterns of organogenesis in thalloid liverworts are unclear. To address this question, we developed a clonal analysis method for use in the thalloid liverwort Marchantia polymorpha, involving random low-frequency induction of a constitutively expressed nuclear-targeted fluorescent protein by dual heat-shock and dexamethasone treatment. M. polymorpha thalli ultimately derive from stem cells in the apical notch, and the lobes predominantly develop from merophytes cleft to the left and right of the apical cell(s). Sector induction in gemmae and subsequent culture occasionally generated fluorescent sectors that bisected thalli along the midrib and were maintained through several bifurcation events, likely reflecting the border between lateral merophytes. Such thallus-bisecting sectors traversed dorsal air chambers and gemma cups, suggesting that these organs arise independently of merophyte cell lineages in response to local positional cues.

Somatic hybridization provides segregating populations for the identification of causative mutations in sterile mutants of the moss Physcomitrella patens.

Forward genetics is now straightforward in the moss Physcomitrella patens, and large mutant populations can be screened relatively easily. However, perturbation of development before the formation of gametes currently leaves no route to gene discovery. Somatic hybridization has previously been used to rescue sterile mutants and to assign P. patens mutations to complementation groups, but the cellular basis of the fusion process could not be monitored, and there was no tractable way to identify causative mutations. Here we use fluorescently tagged lines to generate somatic hybrids between Gransden (Gd) and Villersexel (Vx) strains of P. patens, and show that hybridization produces fertile diploid gametophytes that form phenotypically normal tetraploid sporophytes. Quantification of genetic variation between the two parental strains reveals single nucleotide polymorphisms at a frequency of 1/286 bp. Given that the genetic distinction between Gd and Vx strains exceeds that found between pairs of strains that are commonly used for genetic mapping in other plant species, the spore populations derived from hybrid sporophytes provide suitable material for bulk segregant analysis and gene identification by genome sequencing.

Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling.

Strigolactones (SLs) are key hormonal regulators of flowering plant development and are widely distributed amongst streptophytes. In Arabidopsis, SLs signal via the F-box protein MORE AXILLARY GROWTH2 (MAX2), affecting multiple aspects of development including shoot branching, root architecture and drought tolerance. Previous characterization of a Physcomitrella patens moss mutant with defective SL synthesis supports an ancient role for SLs in land plants, but the origin and evolution of signalling pathway components are unknown. Here we investigate the function of a moss homologue of MAX2, PpMAX2, and characterize its role in SL signalling pathway evolution by genetic analysis. We report that the moss Ppmax2 mutant shows very distinct phenotypes from the moss SL-deficient mutant. In addition, the Ppmax2 mutant remains sensitive to SLs, showing a clear transcriptional SL response in dark conditions, and the response to red light is also altered. These data suggest divergent evolutionary trajectories for SL signalling pathway evolution in mosses and vascular plants. In P. patens, the primary roles for MAX2 are in photomorphogenesis and moss early development rather than in SL response, which may require other, as yet unidentified, factors.

Auxin transport in the evolution of branching forms.

Contents 545 I. 545 II. 546 III. 546 IV. 548 V. 548 VI. 549 VII. 549 Acknowledgements 549 References 549 SUMMARY: Branching is one of the most striking aspects of land plant architecture, affecting resource acquisition and yield. Polar auxin transport by PIN proteins is a primary determinant of flowering plant branching patterns regulating both branch initiation and branch outgrowth. Several lines of experimental evidence suggest that PIN-mediated polar auxin transport is a conserved regulator of branching in vascular plant sporophytes. However, the mechanisms of branching and auxin transport and relationships between the two are not well known outside the flowering plants, and the paradigm for PIN-regulated branching in flowering plants does not fit bryophyte gametophytes. The evidence reviewed here suggests that divergent auxin transport routes contributed to the diversification of branching forms in distinct land plant lineages.

Multiple innovations underpinned branching form diversification in mosses.

Broad-scale evolutionary comparisons have shown that branching forms arose by convergence in vascular plants and bryophytes, but the trajectory of branching form diversification in bryophytes is unclear. Mosses are the most species-rich bryophyte lineage and two sub-groups are circumscribed by alternative reproductive organ placements. In one, reproductive organs form apically, terminating growth of the primary shoot (gametophore) axis. In the other, reproductive organs develop on very short lateral branches. A switch from apical to lateral reproductive organ development is proposed to have primed branching form diversification. Moss gametophores have modular development and each module develops from a single apical cell. Here we define the architectures of 175 mosses by the number of module classes, branching patterns and the pattern in which similar modules repeat. Using ancestral character state reconstruction we identify two stages of architectural diversification. During a first stage there were sequential changes in the module repetition pattern, reproductive organ position, branching pattern and the number of module classes. During a second stage, vegetative changes occurred independently of reproductive fate. The results pinpoint the nature of developmental change priming branching form diversification in mosses and provide a framework for mechanistic studies of architectural diversification.

Paralogous radiations of PIN proteins with multiple origins of noncanonical PIN structure.

The plant hormone auxin is a conserved regulator of development which has been implicated in the generation of morphological novelty. PIN-FORMED1 (PIN) auxin efflux carriers are central to auxin function by regulating its distribution. PIN family members have divergent structures and cellular localizations, but the origin and evolutionary significance of this variation is unresolved. To characterize PIN family evolution, we have undertaken phylogenetic and structural analyses with a massive increase in taxon sampling over previous studies. Our phylogeny shows that following the divergence of the bryophyte and lycophyte lineages, two deep duplication events gave rise to three distinct lineages of PIN proteins in euphyllophytes. Subsequent independent radiations within each of these lineages were taxonomically asymmetric, giving rise to at least 21 clades of PIN proteins, of which 15 are revealed here for the first time. Although most PIN protein clades share a conserved canonical structure with a modular central loop domain, a small number of noncanonical clades dispersed across the phylogeny have highly divergent protein structure. We propose that PIN proteins underwent sub- and neofunctionalization with substantial modification to protein structure throughout plant evolution. Our results have important implications for plant evolution as they suggest that structurally divergent PIN proteins that arose in paralogous radiations contributed to the convergent evolution of organ systems in different land plant lineages.

CLE peptides act via the receptor-like kinase CRINKLY 4 in Physcomitrium patens gametophore development.

The CLAVATA pathway plays a key role in the regulation of multicellular shoot and root meristems in flowering plants. In Arabidopsis, CLAVATA 3-like signaling peptides (CLEs) act via receptor-like kinases CLAVATA 1 and CRINKLY 4 (CR4). In the moss Physcomitrium patens, PpCLAVATA and PpCR4 were previously studied independently and shown to play conserved roles in the regulation of cell proliferation and differentiation. The plant calpain DEFECTIVE KERNEL 1 (DEK1) has been identified as another key regulator of cell division and cell fate in vascular plants and bryophytes. The functional interaction between CLAVATA, CR4, and DEK1 remains unknown. Here, we show that P. patens crinkly4 and dek1 mutants respond differently to CLE peptide treatments suggesting their distinct roles in the CLAVATA pathway. Reduced CLAVATA-mediated suppression of leafy shoot growth in Δcr4 mutants indicates that PpCR4 is involved in CLV3p perception, most likely as a receptor. The CLV3p strongly suppressed leaf vein development in Δcr4 mutants, suggesting that other receptors are involved in these processes and indicating a potential role of PpCR4 in organ sensitization to CLEs.

A modified method to analyse cell proliferation using EdU labelling in large insect brains.

The study of neurogenesis is critical to understanding of the evolution of nervous systems. Within invertebrates, this process has been extensively studied in Drosophila melanogaster, which is the predominant model thanks to the availability of advanced genetic tools. However, insect nervous systems are extremely diverse, and by studying a range of taxa we can gain additional information about how nervous systems and their development evolve. One example of the high diversity of insect nervous system diversity is provided by the mushroom bodies. Mushroom bodies have critical roles in learning and memory and vary dramatically across species in relative size and the type(s) of sensory information they process. Heliconiini butterflies provide a useful snapshot of this diversity within a closely related clade. Within Heliconiini, the genus Heliconius contains species where mushroom bodies are 3-4 times larger than other closely related genera, relative to the rest of the brain. This variation in size is largely explained by increases in the number of Kenyon cells, the intrinsic neurons which form the mushroom body. Hence, variation in mushroom body size is the product of changes in cell proliferation during Kenyon cell neurogenesis. Studying this variation requires adapting labelling techniques for use in less commonly studied organisms, as methods developed for common laboratory insects often do not work. Here, we present a modified protocol for EdU staining to examine neurogenesis in large-brained insects, using Heliconiini butterflies as our primary case, but also demonstrating applicability to cockroaches, another large-brained insect.

Independent recruitment of a conserved developmental mechanism during leaf evolution.

Vascular plants evolved in the Middle to Late Silurian period, about 420 million years ago. The fossil record indicates that these primitive plants had branched stems with sporangia but no leaves. Leaf-like lateral outgrowths subsequently evolved on at least two independent occasions. In extant plants, these events are represented by microphyllous leaves in lycophytes (clubmosses, spikemosses and quillworts) and megaphyllous leaves in euphyllophytes (ferns, gymnosperms and angiosperms). Our current understanding of how leaves develop is restricted to processes that operate during megaphyll formation. Because microphylls and megaphylls evolved independently, different mechanisms might be required for leaf formation. Here we show that this is not so. Gene expression data from a microphyllous lycophyte, phylogenetic analyses, and a cross-species complementation experiment all show that a common developmental mechanism can underpin both microphyll and megaphyll formation. We propose that this mechanism might have operated originally in the context of primitive plant apices to facilitate bifurcation. Recruitment of this pathway to form leaves occurred independently and in parallel in different plant lineages.

The evolutionary emergence of land plants.

There can be no doubt that early land plant evolution transformed the planet but, until recently, how and when this was achieved was unclear. Coincidence in the first appearance of land plant fossils and formative shifts in atmospheric oxygen and CO2 are an artefact of the paucity of earlier terrestrial rocks. Disentangling the timing of land plant bodyplan assembly and its impact on global biogeochemical cycles has been precluded by uncertainty concerning the relationships of bryophytes to one another and to the tracheophytes, as well as the timescale over which these events unfolded. New genome and transcriptome sequencing projects, combined with the application of sophisticated phylogenomic modelling methods, have yielded increasing support for the Setaphyta clade of liverworts and mosses, within monophyletic bryophytes. We consider the evolution of anatomy, genes, genomes and of development within this phylogenetic context, concluding that many vascular plant (tracheophytes) novelties were already present in a comparatively complex last common ancestor of living land plants (embryophytes). Molecular clock analyses indicate that embryophytes emerged in a mid-Cambrian to early Ordovician interval, compatible with hypotheses on their role as geoengineers, precipitating early Palaeozoic glaciations.

A conserved mechanism for nitrile metabolism in bacteria and plants.

Pseudomonas fluorescens SBW25 is a plant growth-promoting bacterium that efficiently colonizes the leaf surfaces and rhizosphere of a range of plants. Previous studies have identified a putative plant-induced nitrilase gene (pinA) in P. fluorescens SBW25 that is expressed in the rhizosphere of sugar beet plants. Nitrilase enzymes have been characterised in plants, bacteria and fungi and are thought to be important in detoxification of nitriles, utilisation of nitrogen and synthesis of plant hormones. We reveal that pinA is a NIT4-type nitrilase that catalyses the hydrolysis of beta-cyano-L-alanine, a nitrile common in the plant environment and an intermediate in the cyanide detoxification pathway in plants. In plants cyanide is converted to beta-cyano-L-alanine, which is subsequently detoxified to aspartic acid and ammonia by NIT4. In P. fluorescens SBW25 pinA is induced in the presence of beta-cyano-L-alanine, and the beta-cyano-L-alanine precursors cyanide and cysteine. pinA allows P. fluorescens SBW25 to use beta-cyano-L-alanine as a nitrogen source and to tolerate toxic concentrations of this nitrile. In addition, pinA is shown to complement a NIT4 mutation in Arabidopsis thaliana, enabling plants to grow in concentrations of beta-cyano-L-alanine that would otherwise prove lethal. Interestingly, over-expression of pinA in wild-type A. thaliana not only resulted in increased growth in high concentrations of beta-cyano-L-alanine, but also resulted in increased root elongation in the absence of exogenous beta-cyano-L-alanine, demonstrating that beta-cyano-L-alanine nitrilase activity can have a significant effect on root physiology and root development.

Meiosis in flowering plants and other green organisms.

Sexual eukaryotes generate gametes using a specialized cell division called meiosis that serves both to halve the number of chromosomes and to reshuffle genetic variation present in the parent. The nature and mechanism of the meiotic cell division in plants and its effect on genetic variation are reviewed here. As flowers are the site of meiosis and fertilization in angiosperms, meiotic control will be considered within this developmental context. Finally, we review what is known about the control of meiosis in green algae and non-flowering land plants and discuss evolutionary transitions relating to meiosis that have occurred in the lineages giving rise to the angiosperms.

Phylogenomic Evidence for the Monophyly of Bryophytes and the Reductive Evolution of Stomata.

The origin of land plants was accompanied by new adaptations to life on land, including the evolution of stomata-pores on the surface of plants that regulate gas exchange. The genes that underpin the development and function of stomata have been extensively studied in model angiosperms, such as Arabidopsis. However, little is known about stomata in bryophytes, and their evolutionary origins and ancestral function remain poorly understood. Here, we resolve the position of bryophytes in the land plant tree and investigate the evolutionary origins of genes that specify stomatal development and function. Our analyses recover bryophyte monophyly and demonstrate that the guard cell toolkit is more ancient than has been appreciated previously. We show that a range of core guard cell genes, including SPCH/MUTE, SMF, and FAMA, map back to the common ancestor of embryophytes or even earlier. These analyses suggest that the first embryophytes possessed stomata that were more sophisticated than previously envisioned and that the stomata of bryophytes have undergone reductive evolution, including their complete loss from liverworts.

Anthoceros genomes illuminate the origin of land plants and the unique biology of hornworts.

Hornworts comprise a bryophyte lineage that diverged from other extant land plants >400 million years ago and bears unique biological features, including a distinct sporophyte architecture, cyanobacterial symbiosis and a pyrenoid-based carbon-concentrating mechanism (CCM). Here, we provide three high-quality genomes of Anthoceros hornworts. Phylogenomic analyses place hornworts as a sister clade to liverworts plus mosses with high support. The Anthoceros genomes lack repeat-dense centromeres as well as whole-genome duplication, and contain a limited transcription factor repertoire. Several genes involved in angiosperm meristem and stomatal function are conserved in Anthoceros and upregulated during sporophyte development, suggesting possible homologies at the genetic level. We identified candidate genes involved in cyanobacterial symbiosis and found that LCIB, a Chlamydomonas CCM gene, is present in hornworts but absent in other plant lineages, implying a possible conserved role in CCM function. We anticipate that these hornwort genomes will serve as essential references for future hornwort research and comparative studies across land plants.