A fundamental developmental transition in Physcomitrium patens is regulated by evolutionarily conserved mechanisms.

One of the most defining moments in history was the colonization of land by plants approximately 470 million years ago. The transition from water to land was accompanied by significant changes in the plant body plan, from those than resembled filamentous representatives of the charophytes, the sister group to land plants, to those that were morphologically complex and capable of colonizing harsher habitats. The moss Physcomitrium patens (also known as Physcomitrella patens) is an extant representative of the bryophytes, the earliest land plant lineage. The protonema of P. patens emerges from spores from a chloronemal initial cell, which can divide to self-renew to produce filaments of chloronemal cells. A chloronemal initial cell can differentiate into a caulonemal initial cell, which can divide and self-renew to produce filaments of caulonemal cells, which branch extensively and give rise to three-dimensional shoots. The process by which a chloronemal initial cell differentiates into a caulonemal initial cell is tightly regulated by auxin-induced remodeling of the actin cytoskeleton. Studies have revealed that the genetic mechanisms underpinning this transition also regulate tip growth and differentiation in diverse plant taxa. This review summarizes the known cellular and molecular mechanisms underpinning the chloronema to caulonema transition in P. patens.

Three-dimensional growth: a developmental innovation that facilitated plant terrestrialization.

One of the most transformative events in the history of life on earth was the transition of plants from water to land approximately 470 million years ago. Within the Charophyte green algae, the closest living relatives of land plants, body plans have evolved from those that comprise simple unicells to those that are morphologically complex, large and multicellular. The Charophytes developed these broad ranging body plans by exploiting a range of one-dimensional and two-dimensional growth strategies to produce filaments, mats and branches. When plants were confronted with harsh conditions on land, they were required to make significant changes to the way they shaped their body plans. One of the fundamental developmental transitions that occurred was the evolution of three-dimensional growth and the acquisition of apical cells with three or more cutting faces. Plants subsequently developed a range of morphological adaptations (e.g. vasculature, roots, flowers, seeds) that enabled them to colonise progressively drier environments. 3D apical growth also evolved convergently in the brown algae, completely independently of the green lineage. This review summarises the evolving developmental complexities observed in the early divergent Charophytes all the way through to the earliest conquerors of land, and investigates 3D apical growth in the brown algae.

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.

Nonreciprocal complementation of KNOX gene function in land plants.

Class I KNOTTED-LIKE HOMEOBOX (KNOX) proteins regulate development of the multicellular diploid sporophyte in both mosses and flowering plants; however, the morphological context in which they function differs. In order to determine how Class I KNOX function was modified as land plants evolved, phylogenetic analyses and cross-species complementation assays were performed. Our data reveal that a duplication within the charophyte sister group to land plants led to distinct Class I and Class II KNOX gene families. Subsequently, Class I sequences diverged substantially in the nonvascular bryophyte groups (liverworts, mosses and hornworts), with moss sequences being most similar to those in vascular plants. Despite this similarity, moss mutants were not complemented by vascular plant KNOX genes. Conversely, the Arabidopsis brevipedicellus (bp-9) mutant was complemented by the PpMKN2 gene from the moss Physcomitrella patens. Lycophyte KNOX genes also complemented bp-9 whereas fern genes only partially complemented the mutant. This lycophyte/fern distinction is mirrored in the phylogeny of KNOX-interacting BELL proteins, in that a gene duplication occurred after divergence of the two groups. Together, our results imply that the moss MKN2 protein can function in a broader developmental context than vascular plant KNOX proteins, the narrower scope having evolved progressively as lycophytes, ferns and flowering plants diverged.

An ancient and conserved function for Armadillo-related proteins in the control of spore and seed germination by abscisic acid.

Armadillo-related proteins regulate development throughout eukaryotic kingdoms. In the flowering plant Arabidopsis thaliana, Armadillo-related ARABIDILLO proteins promote multicellular root branching. ARABIDILLO homologues exist throughout land plants, including early-diverging species lacking true roots, suggesting that early-evolving ARABIDILLOs had additional biological roles. Here we investigated, using molecular genetics, the conservation and diversification of ARABIDILLO protein function in plants separated by c. 450 million years of evolution. We demonstrate that ARABIDILLO homologues in the moss Physcomitrella patens regulate a previously undiscovered inhibitory effect of abscisic acid (ABA) on spore germination. Furthermore, we show that A. thaliana ARABIDILLOs function similarly during seed germination. Early-diverging ARABIDILLO homologues from both P. patens and the lycophyte Selaginella moellendorffii can substitute for ARABIDILLO function during A. thaliana root development and seed germination. We conclude that (1) ABA was co-opted early in plant evolution to regulate functionally analogous processes in spore- and seed-producing plants and (2) plant ARABIDILLO germination functions were co-opted early into both gametophyte and sporophyte, with a specific rooting function evolving later in the land plant lineage.

The decision to germinate is regulated by divergent molecular networks in spores and seeds.

Dispersal is a key step in land plant life cycles, usually via formation of spores or seeds. Regulation of spore- or seed-germination allows control over the timing of transition from one generation to the next, enabling plant dispersal. A combination of environmental and genetic factors determines when seed germination occurs. Endogenous hormones mediate this decision in response to the environment. Less is known about how spore germination is controlled in earlier-evolving nonseed plants. Here, we present an in-depth analysis of the environmental and hormonal regulation of spore germination in the model bryophyte Physcomitrella patens (Aphanoregma patens). Our data suggest that the environmental signals regulating germination are conserved, but also that downstream hormone integration pathways mediating these responses in seeds were acquired after the evolution of the bryophyte lineage. Moreover, the role of abscisic acid and diterpenes (gibberellins) in germination assumed much greater importance as land plant evolution progressed. We conclude that the endogenous hormone signalling networks mediating germination in response to the environment may have evolved independently in spores and seeds. This paves the way for future research about how the mechanisms of plant dispersal on land evolved.

AtMYB93 is a novel negative regulator of lateral root development in Arabidopsis.

Plant root system plasticity is critical for survival in changing environmental conditions. One important aspect of root architecture is lateral root development, a complex process regulated by hormone, environmental and protein signalling pathways. Here we show, using molecular genetic approaches, that the MYB transcription factor AtMYB93 is a novel negative regulator of lateral root development in Arabidopsis. We identify AtMYB93 as an interaction partner of the lateral-root-promoting ARABIDILLO proteins. Atmyb93 mutants have faster lateral root developmental progression and enhanced lateral root densities, while AtMYB93-overexpressing lines display the opposite phenotype. AtMYB93 is expressed strongly, specifically and transiently in the endodermal cells overlying early lateral root primordia and is additionally induced by auxin in the basal meristem of the primary root. Furthermore, Atmyb93 mutant lateral root development is insensitive to auxin, indicating that AtMYB93 is required for normal auxin responses during lateral root development. We propose that AtMYB93 is part of a novel auxin-induced negative feedback loop stimulated in a select few endodermal cells early during lateral root development, ensuring that lateral roots only develop when absolutely required. Putative AtMYB93 homologues are detected throughout flowering plants and represent promising targets for manipulating root systems in diverse crop species.

CURLY LEAF is required for the auxin-dependent regulation of 3-dimensional growth specification in Physcomitrium patens.

The no gametophores 4 ( nog4-R ) mutant cannot make the transition from 2-dimensional (2D) to 3-dimensional (3D) growth in Physcomitrium patens and forms side branch initials that are largely fated to become sporophyte-like structures. We describe the three different developmental trajectories adopted by the nog4-R mutant, all of which result in indeterminate growth and defects in cell division plane orientation. A candidate gene approach confirmed that the causative mutation resided in the CURLY LEAF gene, and we highlight a previously uncharacterized role for CURLY LEAF in maintaining auxin homeostasis in P. patens .

ARABIDILLO gene homologues in basal land plants: species-specific gene duplication and likely functional redundancy.

ARABIDILLO proteins regulate multicellular root development in Arabidopsis thaliana. Conserved ARABIDILLO homologues are present throughout land plants, even in early-evolving plants that do not possess complex root architecture, suggesting that ARABIDILLO genes have additional functions. Here, we have cloned and characterised ARABIDILLO gene homologues from two early-evolving land plants, the bryophyte Physcomitrella patens and the lycophyte Selaginella moellendorffii. We show that two of the PHYSCODILLO genes (PHYSCODILLO1A and -1B) exist as a tail-to-tail tandem array of two almost identical 12 kb sequences, while a third related gene (PHYSCODILLO2) is located elsewhere in the Physcomitrella genome. Physcomitrella possesses a very low percentage of tandemly arrayed genes compared with the later-evolving plants whose genomes have been sequenced to date. Thus, PHYSCODILLO1A and -1B genes represent a relatively unusual gene arrangement. PHYSCODILLO promoters are active largely in the haploid gametophyte, with additional activity at the foot of the sporophyte. The pattern of promoter activity is uniform in filamentous and leafy tissues, suggesting pleiotropic gene functions and likely functional redundancy: the latter possibility is confirmed by the lack of discernible phenotype in a physcodillo2 deletion mutant. Interestingly, the pattern of PHYSCODILLO promoter activity in female reproductive organs is strikingly similar to that of an Arabidopsis homologue, suggesting co-option of some PHYSCODILLO functions or regulation into both the sporophyte and gametophyte. In conclusion, our work identifies and characterises some of the earliest-evolving land plant ARABIDILLO homologues. We confirm that all land plant ARABIDILLO genes arose from a single common ancestor and suggest that PHYSCODILLO proteins have novel and pleiotropic functions, some of which may be conserved in later-evolving plants.

Unravelling 3D growth in the moss Physcomitrium patens.

The colonization of land by plants, and the greening of the terrestrial biosphere, was one of the most important events in the history of life on Earth. The transition of plants from water to land was accompanied, and largely facilitated, by the acquisition of apical cells with three or more cutting faces (3D growth). This enabled plants to develop the morphological characteristics required to survive and reproduce effectively on land and to colonize progressively drier habitats. Most plants develop in such a way that makes genetic studies of 3D growth difficult as the onset of 3D growth is established early during embryo development. On the other hand, in the moss Physcomitrium patens, the onset of 3D growth is preceded by a protracted 2D filamentous phase of the life cycle that can be continuously propagated. P. patens is an ideal model system in which to identify the genetic toolkit underpinning the 2D to 3D growth transition, and this is because 3D growth is not a pre-requisite for survival. Thus, insights into the mechanisms underpinning the formation of apical cells and the subsequent establishment and maintenance of 3D growth have largely been gained through studies in P. patens. This review summarizes the most recently published articles that have provided new and important insights into the mechanisms underpinning 3D growth in P. patens.

ARABIDILLO proteins have a novel and conserved domain structure important for the regulation of their stability.

ARABIDILLO proteins are F-box-Armadillo (ARM) proteins that regulate root branching in Arabidopsis. Many F-box proteins in plants, yeast and mammals are unstable. In plants, the mechanism for this instability has not been fully investigated. Here, we show that a conserved family of plant ARABIDILLO-related proteins has a unique domain structure consisting of an F-box and leucine-rich repeats (LRRs) followed by ARM-repeats. The LRRs are similar to those found in other plant and animal F-box proteins, including cell cycle proteins and hormone receptors. We demonstrate that the LRRs are required for ARABIDILLO1 function in vivo. ARABIDILLO1 protein is unstable: we show that ARABIDILLO1 protein is associated with ubiquitin and is turned over by the proteasome. Both the F-box and LRR regions of ARABIDILLO1 appear to enable this turnover to occur. Application of known lateral root-regulating signals has no effect on ARABIDILLO1 stability. In addition, plants that lack or overexpress ARABIDILLO proteins respond normally to known lateral root-regulating signals. Thus, we suggest that the signal(s) regulating ARABIDILLO stability in vivo may be either highly specific or novel. The structural conservation between ARABIDILLOs and other plant and animal F-box proteins suggests that the stability of other F-box proteins may be controlled by similar mechanisms.

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.

The 2D to 3D growth transition in the moss Physcomitrella patens.

The colonization of land by plants coincided with and was most likely facilitated by the evolution of 3-dimensional (3D) growth. 3D growth is a pivotal feature of all land plants, but most develop in a way that precludes genetic investigation. In the moss Physcomitrella patens, 3D growth (gametophores) is preceded by an extended 2-dimensional (2D) growth phase (protonemata) that can be propagated indefinitely. Studies using P. patens have thus elucidated some of the molecular mechanisms underlying 3D growth regulation. This review summarizes the known molecular mechanisms underlying both the formation of gametophore initial cells and the development of the 3D growth in gametophores.

Genetic Regulation of the 2D to 3D Growth Transition in the Moss Physcomitrella patens.

One of the most important events in the history of life on earth was the colonization of land by plants; this transition coincided with and was most likely enabled by the evolution of 3-dimensional (3D) growth. Today, the diverse morphologies exhibited across the terrestrial biosphere arise from the differential regulation of 3D growth processes during development. In many plants, 3D growth is initiated during the first few divisions of the zygote, and therefore, the genetic basis cannot be dissected because mutants do not survive. However, in mosses, which are representatives of the earliest land plants, 3D shoot growth is preceded by a 2D filamentous phase that can be maintained indefinitely. Here, we used the moss Physcomitrella patens to identify genetic regulators of the 2D to 3D transition. Mutant screens yielded individuals that could only grow in 2D, and through an innovative strategy that combined somatic hybridization with bulk segregant analysis and genome sequencing, the causative mutation was identified in one of them. The NO GAMETOPHORES 1 (NOG1) gene, which encodes a ubiquitin-associated protein, is present only in land plant genomes. In mutants that lack PpNOG1 function, transcripts encoding 3D-promoting PpAPB transcription factors [1] are significantly reduced, and apical initial cells specified for 3D growth are not formed. PpNOG1 acts at the earliest identified stage of the 2D to 3D transition, possibly through degradation of proteins that suppress 3D growth. The acquisition of NOG1 function in land plants could thus have enabled the evolution and development of 3D morphology.

Failure to upregulate Agrp and Orexin in response to activity based anorexia in weight loss vulnerable rats characterized by passive stress coping and prenatal stress experience.

We hypothesize that anorexia nervosa (AN) poses a physiological stress. Therefore, the way an individual copes with stress may affect AN vulnerability. Since prenatal stress (PNS) exposure alters stress responsivity in offspring this may increase their risk of developing AN. We tested this hypothesis using the activity based anorexia (ABA) rat model in control and PNS rats that were characterized by either proactive or passive stress-coping behavior. We found that PNS passively coping rats ate less and lost more weight during the ABA paradigm. Exposure to ABA resulted in higher baseline corticosterone and lower insulin levels in all groups. However, leptin levels were only decreased in rats with a proactive stress-coping style. Similarly, ghrelin levels were increased only in proactively coping ABA rats. Neuropeptide Y (Npy) expression was increased and proopiomelanocortin (Pomc) expression was decreased in all rats exposed to ABA. In contrast, agouti-related peptide (Agrp) and orexin (Hctr) expression were increased in all but the PNS passively coping ABA rats. Furthermore, DNA methylation of the orexin gene was increased after ABA in proactive coping rats and not in passive coping rats. Overall our study suggests that passive PNS rats have innate impairments in leptin and ghrelin in responses to starvation combined with prenatal stress associated impairments in Agrp and orexin expression in response to starvation. These impairments may underlie decreased food intake and associated heightened body weight loss during ABA in the passively coping PNS rats.