Crossroads in the evolution of plant specialized metabolism.

The monophyletic group of embryophytes (land plants) stands out among photosynthetic eukaryotes: they are the sole constituents of the macroscopic flora on land. In their entirety, embryophytes account for the majority of the biomass on land and constitute an astounding biodiversity. What allowed for the massive radiation of this particular lineage? One of the defining features of all land plants is the production of an array of specialized metabolites. The compounds that the specialized metabolic pathways of embryophytes produce have diverse functions, ranging from superabundant structural polymers and compounds that ward off abiotic and biotic challenges, to signaling molecules whose abundance is measured at the nanomolar scale. These specialized metabolites govern the growth, development, and physiology of land plants-including their response to the environment. Hence, specialized metabolites define the biology of land plants as we know it. And they were likely a foundation for their success. It is thus intriguing to find that the closest algal relatives of land plants, freshwater organisms from the grade of streptophyte algae, possess homologs for key enzymes of specialized metabolic pathways known from land plants. Indeed, some studies suggest that signature metabolites emerging from these pathways can be found in streptophyte algae. Here we synthesize the current understanding of which routes of the specialized metabolism of embryophytes can be traced to a time before plants had conquered land.

Constitutive activation of ABA receptors in Arabidopsis reveals unique regulatory circuitries.

Abscisic acid (ABA) is best known for regulating the responses to abiotic stressors. Thus, applications of ABA signaling pathways are considered promising targets for securing yield under stress. ABA levels rise in response to abiotic stress, mounting physiological and metabolic responses that promote plant survival under unfavorable conditions. ABA elicits its effects by binding to a family of soluble receptors found in monomeric and dimeric states, differing in their affinity to ABA and co-receptors. However, the in vivo significance of the biochemical differences between these receptors remains unclear. We took a gain-of-function approach to study receptor-specific functionality. First, we introduced activating mutations that enforce active ABA-bound receptor conformation. We then transformed Arabidopsis ABA-deficient mutants with the constitutive receptors and monitored suppression of the ABA deficiency phenotype. Our findings suggest that PYL4 and PYL5, monomeric ABA receptors, have differential activity in regulating transpiration and transcription of ABA biosynthesis and stress response genes. Through genetic and metabolic data, we demonstrate that PYR1, but not PYL5, is sufficient to activate the ABA positive feedback mechanism. We propose that ABA signaling - from perception to response - flows differently when triggered by different PYLs, due to tissue and transcription barriers, thus resulting in distinct circuitries.

Phylogeny and evolution of streptophyte algae.

The Streptophyta emerged about a billion years ago. Nowadays, this branch of the green lineage is most famous for one of its clades, the land plants (Embryophyta). Although Embryophyta make up the major share of species numbers in Streptophyta, there is a diversity of probably >5000 species of streptophyte algae that form a paraphyletic grade next to land plants. Here, we focus on the deep divergences that gave rise to the diversity of streptophytes, hence particularly on the streptophyte algae. Phylogenomic efforts have not only clarified the position of streptophyte algae relative to land plants, but recent efforts have also begun to unravel the relationships and major radiations within streptophyte algal diversity. We illustrate how new phylogenomic perspectives have changed our view on the evolutionary emergence of key traits, such as intricate signalling networks that are intertwined with multicellular growth and the chemodiverse hotbed from which they emerged. These traits are key for the biology of land plants but were bequeathed from their algal progenitors.

Divergent responses in desiccation experiments in two ecophysiologically different Zygnematophyceae.

Water scarcity can be considered a major stressor on land, with desiccation being its most extreme form. Land plants have found two different solutions to this challenge: avoidance and tolerance. The closest algal relatives to land plants, the Zygnematophyceae, use the latter, and how this is realized is of great interest for our understanding of the conquest of land. Here, we worked with two representatives of the Zygnematophyceae, Zygnema circumcarinatum SAG 698-1b and Mesotaenium endlicherianum SAG 12.97, who differ in habitats and drought resilience. We challenged both algal species with severe desiccation in a laboratory setup until photosynthesis ceased, followed by a recovery period. We assessed their morphological, photophysiological, and transcriptomic responses. Our data pinpoint global differential gene expression patterns that speak of conserved responses, from calcium-mediated signaling to the adjustment of plastid biology, cell envelopes, and amino acid pathways, between Zygnematophyceae and land plants despite their strong ecophysiological divergence. The main difference between the two species appears to rest in a readjustment of the photobiology of Zygnema, while Mesotaenium experiences stress beyond a tipping point.

The evolution of the phenylpropanoid pathway entailed pronounced radiations and divergences of enzyme families.

Land plants constantly respond to fluctuations in their environment. Part of their response is the production of a diverse repertoire of specialized metabolites. One of the foremost sources for metabolites relevant to environmental responses is the phenylpropanoid pathway, which was long thought to be a land-plant-specific adaptation shaped by selective forces in the terrestrial habitat. Recent data have, however, revealed that streptophyte algae, the algal relatives of land plants, have candidates for the genetic toolkit for phenylpropanoid biosynthesis and produce phenylpropanoid-derived metabolites. Using phylogenetic and sequence analyses, we here show that the enzyme families that orchestrate pivotal steps in phenylpropanoid biosynthesis have independently undergone pronounced radiations and divergence in multiple lineages of major groups of land plants; sister to many of these radiated gene families are streptophyte algal candidates for these enzymes. These radiations suggest a high evolutionary versatility in the enzyme families involved in the phenylpropanoid-derived metabolism across embryophytes. We suggest that this versatility likely translates into functional divergence, and may explain the key to one of the defining traits of embryophytes: a rich specialized metabolism.

Unexpected cryptic species among streptophyte algae most distant to land plants.

Streptophytes are one of the major groups of the green lineage (Chloroplastida or Viridiplantae). During one billion years of evolution, streptophytes have radiated into an astounding diversity of uni- and multicellular green algae as well as land plants. Most divergent from land plants is a clade formed by Mesostigmatophyceae, Spirotaenia spp. and Chlorokybophyceae. All three lineages are species-poor and the Chlorokybophyceae consist of a single described species, Chlorokybus atmophyticus. In this study, we used phylogenomic analyses to shed light into the diversity within Chlorokybus using a sampling of isolates across its known distribution. We uncovered a consistent deep genetic structure within the Chlorokybus isolates, which prompted us to formally extend the Chlorokybophyceae by describing four new species. Gene expression differences among Chlorokybus species suggest certain constitutive variability that might influence their response to environmental factors. Failure to account for this diversity can hamper comparative genomic studies aiming to understand the evolution of stress response across streptophytes. Our data highlight that future studies on the evolution of plant form and function can tap into an unknown diversity at key deep branches of the streptophytes.

Evo-physio: on stress responses and the earliest land plants.

Embryophytes (land plants) can be found in almost any habitat on the Earth's surface. All of this ecologically diverse embryophytic flora arose from algae through a singular evolutionary event. Traits that were, by their nature, indispensable for the singular conquest of land by plants were those that are key for overcoming terrestrial stressors. Not surprisingly, the biology of land plant cells is shaped by a core signaling network that connects environmental cues, such as stressors, to the appropriate responses-which, thus, modulate growth and physiology. When did this network emerge? Was it already present when plant terrestrialization was in its infancy? A comparative approach between land plants and their algal relatives, the streptophyte algae, allows us to tackle such questions and resolve parts of the biology of the earliest land plants. Exploring the biology of the earliest land plants might shed light on exactly how they overcame the challenges of terrestrialization. Here, we outline the approaches and rationale underlying comparative analyses towards inferring the genetic toolkit for the stress response that aided the earliest land plants in their conquest of land.

Phylogenomic insights into the first multicellular streptophyte.

Streptophytes are best known as the clade containing the teeming diversity of embryophytes (land plants).1,2,3,4 Next to embryophytes are however a range of freshwater and terrestrial algae that bear important information on the emergence of key traits of land plants. Among these, the Klebsormidiophyceae stand out. Thriving in diverse environments-from mundane (ubiquitous occurrence on tree barks and rocks) to extreme (from the Atacama Desert to the Antarctic)-Klebsormidiophyceae can exhibit filamentous body plans and display remarkable resilience as colonizers of terrestrial habitats.5,6 Currently, the lack of a robust phylogenetic framework for the Klebsormidiophyceae hampers our understanding of the evolutionary history of these key traits. Here, we conducted a phylogenomic analysis utilizing advanced models that can counteract systematic biases. We sequenced 24 new transcriptomes of Klebsormidiophyceae and combined them with 14 previously published genomic and transcriptomic datasets. Using an analysis built on 845 loci and sophisticated mixture models, we establish a phylogenomic framework, dividing the six distinct genera of Klebsormidiophyceae in a novel three-order system, with a deep divergence more than 830 million years ago. Our reconstructions of ancestral states suggest (1) an evolutionary history of multiple transitions between terrestrial-aquatic habitats, with stem Klebsormidiales having conquered land earlier than embryophytes, and (2) that the body plan of the last common ancestor of Klebsormidiophyceae was multicellular, with a high probability that it was filamentous whereas the sarcinoids and unicells in Klebsormidiophyceae are likely derived states. We provide evidence that the first multicellular streptophytes likely lived about a billion years ago.

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.

Controlling the kinetic chain length of the crosslinks in photo-polymerized biodegradable networks.

Biodegradable polymer networks were prepared by photo-initiated radical polymerization of methacrylate functionalized poly(D,L-lactide) oligomers. The kinetic chains formed in this radical polymerization are the multifunctional crosslinks of the networks. These chains are carbon-carbon chains that remain after degradation. If their molecular weight is too high these poly(methacrylic acid) chains can not be excreted by the kidneys. The effect of the photo-initiator concentration and the addition of 2-mercaptoethanol as a chain transfer agent on the molecular weight of the kinetic chains was investigated. It was found that both increasing the initiator concentration and adding 2-mercaptoethanol decrease the kinetic chain length. However, the effect of adding 2-mercaptoethanol was much larger. Some network properties such as the glass transition temperature and the swelling ratio in acetone are affected when the kinetic chain length is decreased.

Environmental gradients reveal stress hubs pre-dating plant terrestrialization.

Plant terrestrialization brought forth the land plants (embryophytes). Embryophytes account for most of the biomass on land and evolved from streptophyte algae in a singular event. Recent advances have unravelled the first full genomes of the closest algal relatives of land plants; among the first such species was Mesotaenium endlicherianum. Here we used fine-combed RNA sequencing in tandem with a photophysiological assessment on Mesotaenium exposed to a continuous range of temperature and light cues. Our data establish a grid of 42 different conditions, resulting in 128 transcriptomes and ~1.5 Tbp (~9.9 billion reads) of data to study the combinatory effects of stress response using clustering along gradients. Mesotaenium shares with land plants major hubs in genetic networks underpinning stress response and acclimation. Our data suggest that lipid droplet formation and plastid and cell wall-derived signals have denominated molecular programmes since more than 600 million years of streptophyte evolution-before plants made their first steps on land.

Chromosome-level genomes of multicellular algal sisters to land plants illuminate signaling network evolution.

The filamentous and unicellular algae of the class Zygnematophyceae are the closest algal relatives of land plants. Inferring the properties of the last common ancestor shared by these algae and land plants allows us to identify decisive traits that enabled the conquest of land by plants. We sequenced four genomes of filamentous Zygnematophyceae (three strains of Zygnema circumcarinatum and one strain of Z. cylindricum) and generated chromosome-scale assemblies for all strains of the emerging model system Z. circumcarinatum. Comparative genomic analyses reveal expanded genes for signaling cascades, environmental response, and intracellular trafficking that we associate with multicellularity. Gene family analyses suggest that Zygnematophyceae share all the major enzymes with land plants for cell wall polysaccharide synthesis, degradation, and modifications; most of the enzymes for cell wall innovations, especially for polysaccharide backbone synthesis, were gained more than 700 million years ago. In Zygnematophyceae, these enzyme families expanded, forming co-expressed modules. Transcriptomic profiling of over 19 growth conditions combined with co-expression network analyses uncover cohorts of genes 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.

Submergence of the filamentous Zygnematophyceae Mougeotia induces differential gene expression patterns associated with core metabolism and photosynthesis.

The streptophyte algal class Zygnematophyceae is the closest algal sister lineage to land plants. In nature, Zygnematophyceae can grow in both terrestrial and freshwater habitats and how they do this is an important unanswered question. Here, we studied what happens to the zygnematophyceaen alga Mougeotia sp., which usually occurs in permanent and temporary freshwater bodies, when it is shifted to liquid growth conditions after growth on a solid substrate. Using global differential gene expression profiling, we identified changes in the core metabolism of the organism interlinked with photosynthesis; the latter went hand in hand with measurable impact on the photophysiology as assessed via pulse amplitude modulation (PAM) fluorometry. Our data reveal a pronounced change in the overall physiology of the alga after submergence and pinpoint candidate genes that play a role. These results provide insight into the importance of photophysiological readjustment when filamentous Zygnematophyceae transition between terrestrial and aquatic habitats.

Fumaric acid monoethyl ester-functionalized poly(D,L-lactide)/N-vinyl-2-pyrrolidone resins for the preparation of tissue engineering scaffolds by stereolithography.

Polymer networks were prepared by photocross-linking fumaric acid monoethyl ester (FAME) functionalized, three-armed poly(D,L-lactide) oligomers using N-vinyl-2-pyrrolidone (NVP) as diluent and comonomer. The use of NVP together with FAME-functionalized oligomers resulted in copolymerization at high rates, and networks with gel contents in excess of 90% were obtained. The hydrophilicity of the poly(D,L-lactide) networks increases with increasing amounts of NVP, networks containing 50 wt % of NVP absorbed 40% of water. As the amount of NVP was increased from 30 to 50 wt %, the Young's modulus after equilibration in water decreased from 0.8 to 0.2 GPa, as opposed to an increase from 1.5 to 2.1 GPa in the dry state. Mouse preosteoblasts readily adhered and spread onto all prepared networks. Using stereolithography, porous structures with a well-defined gyroid architecture were prepared from these novel materials. This allows the preparation of tissue engineering scaffolds with optimized pore architecture and tunable material properties.

Photo-crosslinked biodegradable hydrogels prepared from fumaric acid monoethyl ester-functionalized oligomers for protein delivery.

Photo-crosslinkable, fumaric acid monoethyl ester-functionalized triblock oligomers are synthesized and copolymerized with N-vinyl-2-pyrrolidone to form biodegradable photo-crosslinked hydrogels. Poly(ethylene glycol) is used as the middle hydrophilic segment and the hydrophobic segments are based on D,L-lactide, trimethylene carbonate or a mixture of these monomers. Two model proteins, lysozyme and albumin, are incorporated in the hydrogels and their release is studied. The composition of the hydrophobic segments could be used to tune degradation behavior and release rates. Careful optimization of photo-polymerization conditions is needed to limit conjugation of proteins to the hydrogels and protein denaturation.

Photo-crosslinked networks prepared from fumaric acid monoethyl ester-functionalized poly(D,L-lactic acid) oligomers and N-vinyl-2-pyrrolidone for the controlled and sustained release of proteins.

Photo-crosslinked networks were prepared from fumaric acid monoethyl ester-functionalized poly(D,L-lactic acid) oligomers and N-vinyl-2-pyrrolidone. Two model proteins, lysozyme and albumin, were incorporated into the network films as solid particles and their release behavior was studied. By varying the NVP content and macromer molecular weight the degradation behavior and protein release profiles of the prepared networks could be tuned. The more hydrophilic and less densely crosslinked networks released albumin and lysozyme at a faster rate. Although active lysozyme was released from the networks over the complete release period, lysozyme release was often incomplete. This was most likely caused by electrostatic and/or hydrophobic interactions between the protein and the degrading polymer network.

Intraocular degradation behavior of crosslinked and linear poly(trimethylene carbonate) and poly(D,L-lactic acid).

The intraocular degradation behavior of poly(trimethylene carbonate) (PTMC) networks and poly(D,L-lactic acid) (PDLLA) networks and of linear high molecular weight PTMC and PDLLA was evaluated. PTMC is known to degrade by enzymatic surface erosion in vivo, whereas PDLLA degrades by hydrolytic bulk degradation. Rod shaped specimens were implanted in the vitreous of New Zealand white rabbits for 6 or 13 wk. All materials were well tolerated in the rabbit vitreous. The degradation of linear high molecular weight PTMC and PTMC networks was very slow and no significant mass loss was observed within 13 wk. Only some minor signs of macrophage mediated erosion were found. The fact that no significant enzymatic surface erosion occurs can be related to the avascularity of the vitreous and the limited number of cells it contains. PDLLA samples showed more evident signs of degradation. For linear PDLLA significant swelling and a large decrease in molecular weight in time was observed and PDLLA network implants started to lose mass within 13 wk. Of the tested materials, PDLLA networks seem to be most promising for long term degradation controlled intravitreal drug delivery since this material degrades without significant swelling. Furthermore the preparation method of these networks allows easy and efficient incorporation of drugs.

Rapid photo-crosslinking of fumaric acid monoethyl ester-functionalized poly(trimethylene carbonate) oligomers for drug delivery applications.

Photo-crosslinkable, fumaric acid monoethyl ester-functionalized poly(trimethylene carbonate) oligomers were synthesized and copolymerized with N-vinyl pyrrolidone (NVP) and vinyl acetate (VAc) to form biodegradable polymer networks. The copolymerization reactions were much faster than homopolymerization of the fumarate end-groups of the macromers. The hydrophilicity of the networks could by varied by mixing NVP and VAc at different ratios. The prepared network extracts were compatible with NIH 3T3 fibroblasts. Release of vitamin B12, used as a model drug, could be tuned by varying network hydrophilicity and macromer molecular weight. A more hydrophilic and less densely crosslinked network resulted in faster release.