Evolution and diversification of the ACT-like domain associated with plant basic helix-loop-helix transcription factors.

Basic helix-loop-helix (bHLH) proteins are one of the largest families of transcription factor (TF) in eukaryotes, and ~30% of all flowering plants' bHLH TFs contain the aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain at variable distances C-terminal from the bHLH. However, the evolutionary history and functional consequences of the bHLH/ACT-like domain association remain unknown. Here, we show that this domain association is unique to the plantae kingdom with green algae (chlorophytes) harboring a small number of bHLH genes with variable frequency of ACT-like domain's presence. bHLH-associated ACT-like domains form a monophyletic group, indicating a common origin. Indeed, phylogenetic analysis results suggest that the association of ACT-like and bHLH domains occurred early in Plantae by recruitment of an ACT-like domain in a common ancestor with widely distributed ACT DOMAIN REPEAT (ACR) genes by an ancestral bHLH gene. We determined the functional significance of this association by showing that Chlamydomonas reinhardtii ACT-like domains mediate homodimer formation and negatively affect DNA binding of the associated bHLH domains. We show that, while ACT-like domains have experienced faster selection than the associated bHLH domain, their rates of evolution are strongly and positively correlated, suggesting that the evolution of the ACT-like domains was constrained by the bHLH domains. This study proposes an evolutionary trajectory for the association of ACT-like and bHLH domains with the experimental characterization of the functional consequence in the regulation of plant-specific processes, highlighting the impacts of functional domain coevolution.

Every refuge has its price: Ostreobium as a model for understanding how algae can live in rock and stay in business.

Ostreobium is a siphonous green alga in the Bryopsidales (Chlorophyta) that burrows into calcium carbonate (CaCO3) substrates. In this habitat, it lives under environmental conditions unusual for an alga (i.e., low light and low oxygen) and it is a major agent of carbonate reef bioerosion. In coral skeletons, Ostreobium can form conspicuous green bands recognizable by the naked eye and it is thought to contribute to the coral's nutritional needs. With coral reefs in global decline, there is a renewed focus on understanding Ostreobium biology and its roles in the coral holobiont. This review summarizes knowledge on Ostreobium's morphological structure, biodiversity and evolution, photosynthesis, mechanism of bioerosion and its role as a member of the coral holobiont. We discuss the resources available to study Ostreobium biology, lay out some of the uncharted territories in Ostreobium biology and offer perspectives for future research.

Characterization of activating cis-regulatory elements from the histone genes of Chlamydomonas reinhardtii.

Regulation of gene expression in eukaryotes is controlled by cis-regulatory modules (CRMs). A major class of CRMs are enhancers which are composed of activating cis-regulatory elements (CREs) responsible for upregulating transcription. To date, most enhancers and activating CREs have been studied in angiosperms; in contrast, our knowledge about these key regulators of gene expression in green algae is limited. In this study, we aimed at characterizing putative activating CREs/CRMs from the histone genes of the unicellular model alga Chlamydomonas reinhardtii. To test the activity of four candidates, reporter constructs consisting of a tetramerized CRE, an established promoter, and a gene for the mCerulean3 fluorescent protein were incorporated into the nuclear genome of C. reinhardtii, and their activity was quantified by flow cytometry. Two tested candidates, Eupstr and Ehist cons, significantly upregulated gene expression and were characterized in detail. Eupstr, which originates from highly expressed genes of C. reinhardtii, is an orientation-independent CRE capable of activating both the RBCS2 and ß2-tubulin promoters. Ehist cons, which is a CRM from histone genes of angiosperms, upregulates the ß2-tubulin promoter in C. reinhardtii over a distance of at least 1.5 kb. The octamer motif present in Ehist cons was identified in C. reinhardtii and the related green algae Chlamydomonas incerta, Chlamydomonas schloesseri, and Edaphochlamys debaryana, demonstrating its high evolutionary conservation. The results of this investigation expand our knowledge about the regulation of gene expression in green algae. Furthermore, the characterized activating CREs/CRMs can be applied as valuable genetic tools.

Phosphate Limitation Responses in Marine Green Algae Are Linked to Reprogramming of the tRNA Epitranscriptome and Codon Usage Bias.

Marine algae are central to global carbon fixation, and their productivity is dictated largely by resource availability. Reduced nutrient availability is predicted for vast oceanic regions as an outcome of climate change; however, there is much to learn regarding response mechanisms of the tiny picoplankton that thrive in these environments, especially eukaryotic phytoplankton. Here, we investigate responses of the picoeukaryote Micromonas commoda, a green alga found throughout subtropical and tropical oceans. Under shifting phosphate availability scenarios, transcriptomic analyses revealed altered expression of transfer RNA modification enzymes and biased codon usage of transcripts more abundant during phosphate-limiting versus phosphate-replete conditions, consistent with the role of transfer RNA modifications in regulating codon recognition. To associate the observed shift in the expression of the transfer RNA modification enzyme complement with the transfer RNAs encoded by M. commoda, we also determined the transfer RNA repertoire of this alga revealing potential targets of the modification enzymes. Codon usage bias was particularly pronounced in transcripts encoding proteins with direct roles in managing phosphate limitation and photosystem-associated proteins that have ill-characterized putative functions in "light stress." The observed codon usage bias corresponds to a proposed stress response mechanism in which the interplay between stress-induced changes in transfer RNA modifications and skewed codon usage in certain essential response genes drives preferential translation of the encoded proteins. Collectively, we expose a potential underlying mechanism for achieving growth under enhanced nutrient limitation that extends beyond the catalog of up- or downregulated protein-encoding genes to the cell biological controls that underpin acclimation to changing environmental conditions.

Organic compounds drive growth in phytoplankton taxa from different functional groups.

Phytoplankton are usually considered autotrophs, but an increasing number of studies show that many taxa are able also to use organic carbon. Acquiring nutrients and energy from different sources might enable an efficient uptake of required substances and provide a strategy to deal with varying resource availability, especially in highly dynamic ecosystems such as estuaries. In our study, we investigated the effects of 31 organic carbon sources on the growth (proxied by differences in cell counts after 24 h exposure) of 17 phytoplankton strains from the Elbe estuary spanning four functional groups. All of our strains were able to make use of at least 1 and up to 26 organic compounds for growth. Pico-sized green algae such as Mychonastes, as well as the nano-sized green alga Monoraphidium in particular were positively affected by a high variety of substances. Reduced light availability, typically appearing in turbid estuaries and similar habitats, resulted in an overall poorer ability to use organic substances for growth, indicating that organic carbon acquisition was not primarily a strategy to deal with darkness. Our results give further evidence for mixotrophy being a ubiquitous ability of phytoplankton and highlight the importance to consider this trophic strategy in research.

A direct PCR approach with low-biomass insert opens new horizons for molecular sciences on cryptogam communities.

Molecular sequence data have transformed research on cryptogams (e.g., lichens, microalgae, fungi, and symbionts thereof) but methods are still strongly hampered by the small size and intermingled growth of the target organisms, poor cultivability and detrimental effects of their secondary metabolites. Here, we aim to showcase examples on which a modified direct PCR approach for diverse aspects of molecular work on environmental samples concerning biocrusts, biofilms, and cryptogams gives new options for the research community. Unlike traditional approaches, this methodology only requires biomass equivalent to colonies and fragments of 0.2 mm in diameter, which can be picked directly from the environmental sample, and includes a quick DNA lysis followed by a standardized PCR cycle that allows co-cycling of various organisms/target regions in the same run. We demonstrate that this modified method can (i) amplify the most widely used taxonomic gene regions and those used for applied and environmental sciences from single colonies and filaments of free-living cyanobacteria, bryophytes, fungi, and lichens, including their mycobionts, chlorobionts, and cyanobionts from both isolates and in situ material during co-cycling; (ii) act as a tool to confirm that the dominant lichen photobiont was isolated from the original sample; and (iii) optionally remove inhibitory secondary lichen substances. Our results represent examples which highlight the method's potential for future applications covering mycology, phycology, biocrusts, and lichenology, in particular.IMPORTANCECyanobacteria, green algae, lichens, and other cryptogams play crucial roles in complex microbial systems such as biological soil crusts of arid biomes or biofilms in caves. Molecular investigations on environmental samples or isolates of these microorganisms are often hampered by their dense aggregation, small size, or metabolism products which complicate DNA extraction and subsequent PCRs. Our work presents various examples of how a direct DNA extraction and PCR method relying on low biomass inserts can overcome these common problems and discusses additional applications of the workflow including adaptations.

Elemental and macromolecular plasticity of Chlamydomonas reinhardtii (Chlorophyta) in response to resource limitation and growth rate.

With the ongoing differential disruption of the biogeochemical cycles of major elements that are essential for all life (carbon, nitrogen, and phosphorus), organisms are increasingly faced with a heterogenous supply of these elements in nature. Given that photosynthetic primary producers form the base of aquatic food webs, impacts of changed elemental supply on these organisms are particularly important. One way that phytoplankton cope with the differential availability of nutrients is through physiological changes, resulting in plasticity in macromolecular and elemental biomass composition. Here, we assessed how the green alga Chlamydomonas reinhardtii adjusts its macromolecular (e.g., carbohydrates, lipids, and proteins) and elemental (C, N, and P) biomass pools in response to changes in growth rate and the modification of resources (nutrients and light). We observed that Chlamydomonas exhibits considerable plasticity in elemental composition (e.g., molar ratios ranging from 124 to 971 for C:P, 4.5 to 25.9 for C:N, and 15.1 to 61.2 for N:P) under all tested conditions, pointing to the adaptive potential of Chlamydomonas in a changing environment. Exposure to low light modified the elemental and macromolecular composition of cells differently than limitation by nutrients. These observed differences, with potential consequences for higher trophic levels, included smaller cells, shifts in C:N and C:P ratios (due to proportionally greater N and P contents), and differential allocation of C among macromolecular pools (proportionally more lipids than carbohydrates) with different energetic value. However, substantial pools of N and P remained unaccounted for, especially at fast growth, indicating accumulation of N and P in forms we did not measure.

Green Seaweed Caulerpa racemosa as a Novel Non-Small Cell Lung Cancer Inhibitor in Overcoming Tyrosine Kinase Inhibitor Resistance: An Analysis Employing Network Pharmacology, Molecular Docking, and In Vitro Research.

The marine environment provides a rich source of distinct creatures containing potentially revolutionary bioactive chemicals. One of these organisms is Caulerpa racemosa, a type of green algae known as green seaweed, seagrapes, or green caviar. This organism stands out because it has great promise for use in medicine, especially in the study of cancer. Through the utilization of computational modeling (in silico) and cellular laboratory experiments (in vitro), the chemical components included in the green seaweed C. racemosa were effectively analyzed, uncovering its capability to treat non-small cell lung cancer (NSCLC). The study specifically emphasized blocking SRC, STAT3, PIK3CA, MAPK1, EGFR, and JAK1 using molecular docking and in vitro. These proteins play a crucial role in the EGFR Tyrosine Kinase Inhibitor Resistance pathway in NSCLC. The chemical Caulersin (C2) included in C. racemosa extract (CRE) has been identified as a potent and effective agent in fighting against non-small cell lung cancer (NSCLC), both in silico and in vitro. CRE and C2 showed a level of inhibition similar to that of osimertinib (positive control/NSCLC drug).

Impact of copper sulphate treatment on cyanobacterial blooms and subsequent water quality risks.

Control of algal blooms and associated biologically-induced water quality risks in drinking reservoirs is problematic. Copper sulphate (CuSO4) treatment is one intervention that has been utilised for >100 years. Evidence indicates a favourable short-term reduction in Cyanobacterial biomass (e.g. bloom termination), but here we indicate that it may also increase longer-term water quality risk. In 2022, we investigated the impacts of CuSO4 spraying on Cyanobacterial communities and nutrient levels within a drinking water supply reservoir using environmental DNA (eDNA) to assess community shifts, alongside monitoring nutrient fractions, orthophosphate (OP) and total phosphate (TP), post-treatment. CuSO4 application successfully reduced Cyanobacterial abundance, however elimination of Cyanobacteria resulted in a shift in bacterial dominance favouring Planctomycetota throughout the summer and a combination of Actinobacteriota and Verrucomicrobiota, throughout autumn. As Cyanobacterial abundance recovered post-treatment, Cyanobacterial genera demonstrated greater diversity compared to only three Cyanobacterial genera present across samples pre-treatment, and included taxa associated with water quality risk (e.g. taste and odour (T&O) metabolite and toxin producers). The increase in Cyanobacteria post-treatment was attributed to an increase in biologically available nutrients, primarily a significant increase in OP. Overall, findings suggest that the significant shift in biodiversity likely induces a less stable ecosystem with greater plasticity of response to changing environmental and biogeochemical variables. Legacy implications of CuSO4 spraying, in terms of shifts in ecosystem and nutrient balance over time, may have implications for drinking water quality, but importantly also for reservoir management options. As such, the effects of CuSO4 spraying should be considered carefully before consideration as a contender for in-reservoir biological control.

Blue Green Algae.

Blue green algae cyanotoxins have become increasingly more prevalent due to environmental, industrial, and agricultural changes that promote their growth into harmful algal blooms. Animals are usually exposed via water used for drinking or bathing, though specific cases related to equines are very limited. The toxic dose for horses has not been determined, and currently only experimental data in other animals can be relied upon to aid in case interpretation and treatment. Treatment is mostly limited to supportive care, and preventative control methods to limit exposures are more likely to aid in animal health until more research has been performed.

The revealing of a novel lipid transfer protein lineage in green algae.

BACKGROUND: Non-specific lipid transfer proteins (nsLTPs) are a group of small and basic proteins that can bind and transfer various lipid molecules to the apoplastic space. A typical nsLTP carries a conserved architecture termed eight-cysteine motif (8CM), a scaffold of loop-linked helices folding into a hydrophobic cavity for lipids binding. Encoded by a multigene family, nsLTPs are widely distributed in terrestrial plants from bryophytes to angiosperms with dozens of gene members in a single species. Although the nsLTPs in the most primitive plants such as Marchantia already reach 14 members and are divergent enough to form separate groups, so far none have been identified in any species of green algae. RESULTS: By using a refined searching strategy, we identified putative nsLTP genes in more than ten species of green algae as one or two genes per haploid genome but not in red and brown algae. The analyses show that the algal nsLTPs carry unique characteristics, including the extended 8CM spacing, larger molecular mass, lower pI value and multiple introns in a gene, which suggests that they could be a novel nsLTP lineage. Moreover, the results of further investigation on the two Chlamydomonas nsLTPs using transcript and protein assays demonstrated their late zygotic stage expression patterns and the canonical nsLTP properties were also verified, such as the fatty acids binding and proteinase resistance activities. CONCLUSIONS: In conclusion, a novel nsLTP lineage is identified in green algae, which carries some unique sequences and molecular features that are distinguishable from those in land plants. Combined with the results of further examinations of the Chlamydomonas nsLTPs in vitro, possible roles of the algal nsLTPs are also suggested. This study not only reveals the existence of the nsLTPs in green algae but also contributes to facilitating future studies on this enigmatic protein family.

Cryopreservation of two species of the multicellular volvocine green algal genus Astrephomene.

BACKGROUND: Astrephomene is an interesting green algal genus that, together with Volvox, shows convergent evolution of spheroidal multicellular bodies with somatic cells of the colonial or multicellular volvocine lineage. A recent whole-genome analysis of A. gubernaculifera resolved the molecular-genetic basis of such convergent evolution, and two species of Astrephomene were described. However, maintenance of culture strains of Astrephomene requires rapid inoculation of living cultures, and cryopreserved culture strains have not been established in public culture collections. RESULTS: To establish cryopreserved culture strains of two species of Astrephomene, conditions for cryopreservation of the two species were investigated using immature and mature vegetative colonies and two cryoprotectants: N,N-dimethylformamide (DMF) and hydroxyacetone (HA). Rates of cell survival of the A. gubernaculifera or A. perforata strain after two-step cooling and freezing in liquid nitrogen were compared between different concentrations (3 and 6%) of DMF and HA and two types of colonies: immature colonies (small colonies newly released from the parent) and mature colonies (large colonies just before daughter colony formation). The highest rate of survival [11 ± 13% (0.36-33%) by the most probable number (MPN) method] of A. gubernaculifera strain NIES-4017 (established in 2014) was obtained when culture samples of immature colonies were subjected to cryogenic treatment with 6% DMF. In contrast, culture samples of mature colonies subjected to 3% HA cryogenic treatment showed the highest "MPN survival" [5.5 ± 5.9% (0.12-12%)] in A. perforata. Using the optimized cryopreservation conditions for each species, survival after freezing in liquid nitrogen was examined for six other strains of A. gubernaculifera (established from 1962 to 1981) and another A. perforata strain maintained in the Microbial Culture Collection at the National Institute for Environmental Studies (MCC-NIES). We obtained ≥0.1% MPN survival of the A. perforata strain. However, only two of the six strains of A. gubernaculifera showed ≥0.1% MPN survival. By using the optimal cryopreserved conditions obtained for each species, five cryopreserved strains of two species of Astrephomene were established and deposited in the MCC-NIES. CONCLUSIONS: The optimal cryopreservation conditions differed between the two species of Astrephomene. Cryopreservation of long-term-maintained strains of A. gubernaculifera may be difficult; further studies of cryopreservation of these strains are needed.

Hydrocytium expands the phylogenetic, morphological, and genomic diversity of the poorly known green algal order Chaetopeltidales.

PREMISE: Chaetopeltidales is a small, understudied order of the green algal class Chlorophyceae, that is slowly expanding with the occasional discoveries of novel algae. Here we demonstrate that hitherto unrecognized chaetopeltidaleans also exist among previously described but neglected and misclassified species. METHODS: Strain SAG 40.91 of Characium acuminatum, shown by previous preliminary evidence to have affinities with the orders Oedogoniales, Chaetophorales, and Chaetopeltidales (together constituting the OCC clade), was investigated with light and electron microscopy to characterize its morphology and ultrastructure. Sequence assemblies of the organellar and nuclear genomes were obtained and utilized in bioinformatic and phylogenetic analyses to address the phylogenetic position of the alga and its salient genomic features. RESULTS: The characterization of strain SAG 40.91 and a critical literature review led us to reinstate the forgotten genus Hydrocytium A.Braun 1855, with SAG 40.91 representing its type species, Hydrocytium acuminatum. Independent molecular markers converged on placing H. acuminatum as a deeply diverged lineage of the order Chaetopeltidales, formalized as the new family Hydrocytiaceae. Both chloroplast and mitochondrial genomes shared characteristics with other members of Chaetopeltidales and were bloated by repetitive sequences. Notably, the mitochondrial cox2a gene was transferred into the nuclear genome in the H. acuminatum lineage, independently of the same event in Volvocales. The nuclear genome data from H. acuminatum and from another chaetopeltidalean that was reported by others revealed endogenized viral sequences corresponding to novel members of the phylum Nucleocytoviricota. CONCLUSIONS: The resurrected genus Hydrocytium expands the known diversity of chaetopeltidalean algae and provides the first glimpse into their virosphere.

Mitochondrial uniparental inheritance achieved after fertilization challenges the nuclear-cytoplasmic conflict hypothesis for anisogamy evolution.

In eukaryotes, a fundamental phenomenon underlying sexual selection is the evolution of gamete size dimorphism between the sexes (anisogamy) from an ancestral gametic system with gametes of the same size in both mating types (isogamy). The nuclear-cytoplasmic conflict hypothesis has been one of the major theoretical hypotheses for the evolution of anisogamy. It proposes that anisogamy evolved as an adaptation for preventing nuclear-cytoplasmic conflict by minimizing male gamete size to inherit organelles uniparentally. In ulvophycean green algae, biparental inheritance of organelles is observed in isogamous species, as the hypothesis assumes. So we tested the hypothesis by examining whether cytoplasmic inheritance is biparental in Monostroma angicava, a slightly anisogamous ulvophycean that produces large male gametes. We tracked the fates of mitochondria in intraspecific crosses with PCR-RFLP markers. We confirmed that mitochondria are maternally inherited. However, paternal mitochondria enter the zygote, where their DNA can be detected for over 14 days. This indicates that uniparental inheritance is enforced by eliminating paternal mitochondrial DNA in the zygote, rather than by decreasing male gamete size to the minimum. Thus, uniparental cytoplasmic inheritance is achieved by an entirely different mechanism, and is unlikely to drive the evolution of anisogamy in ulvophyceans.

Chloroplast genome evolution and phylogeny of the early-diverging charophycean green algae with a focus on the Klebsormidiophyceae and Streptofilum.

The Klebsormidiophyceae are a class of green microalgae observed globally in both freshwater and terrestrial habitats. Morphology-based classification schemes of this class have been shown to be inadequate due to the simple morphology of these algae, the tendency of morphology to vary in culture versus field conditions, and rampant morphological homoplasy. Molecular studies revealing cryptic diversity have renewed interest in this group. We sequenced the complete chloroplast genomes of a broad series of taxa spanning the known taxonomic breadth of this class. We also sequenced the chloroplast genomes of three strains of Streptofilum, a recently discovered green algal lineage with close affinity to the Klebsormidiophyceae. Our results affirm the previously hypothesized polyphyly of the genus Klebsormidium as well as the polyphyly of the nominal species in this genus, K. flaccidum. Furthermore, plastome sequences strongly support the status of Streptofilum as a distinct, early-diverging lineage of charophytic algae sister to a clade comprising Klebsormidiophyceae plus Phragmoplastophyta. We also uncovered major structural alterations in the chloroplast genomes of species in Klebsormidium that have broad implications regarding the underlying mechanisms of chloroplast genome evolution.

Tuning cationic/anionic dyes sorption from aqueous solution onto green algal biomass for biohydrogen production.

Global water security and energy demands associated with uncontrollable population growth and rapid industrial progress are one of the utmost serious needs dangerously confronting humanity. On account of waste as a wealth strategy; a multifunctional eco-friendly sorbent (MGAP) from green alga was prepared successfully for remediation of cationic/anionic organic dyes and biohydrogen production. The structural and morphological properties of sorbent were systematically scrutinized by a variety of spectral analyses. The loading capacity of MGAP towards rhodamine B (RhB) and methyl orange (MO) dyes was inclusivity inspected under variable experimental conditions. The adsorption kinetics of both dyes onto MGAP was in good agreement with pseudo-second-order theory, whereas adsorption isotherms could fit well with the Langmuir model, with satisfactory loading capacities of 144.92 and 196.04 mg g-1 for RhB and MO molecules, respectively. Moreover, ultra-sonication treatment admirably decreased the sorption equilibrium time from 180.0 min to 30.0 min. Furthermore, spent sorbent was managed particularly for biohydrogen production with a measured yield of 112.89, 116.59, and 128.17 mL-H2/gVS for MGAP, MGAP-MO, and MGAP-RhB, respectively. Overall, the produced MGAP can potentially be offered up as a promising dye scavenger for wastewater remediation and biohydrogen production, thereby fulfilling waste management and circular economy.

Enhanced Wound Healing Potential of Spirulina platensis Nanophytosomes: Metabolomic Profiling, Molecular Networking, and Modulation of HMGB-1 in an Excisional Wound Rat Model.

Excisional wounds are considered one of the most common physical injuries. This study aims to test the effect of a nanophytosomal formulation loaded with a dried hydroalcoholic extract of S. platensis on promoting excisional wound healing. The Spirulina platensis nanophytosomal formulation (SPNP) containing 100 mg PC and 50 mg CH exhibited optimum physicochemical characteristics regarding particle size (598.40 ± 9.68 nm), zeta potential (-19.8 ± 0.49 mV), entrapment efficiency (62.76 ± 1.75%), and Q6h (74.00 ± 1.90%). It was selected to prepare an HPMC gel (SPNP-gel). Through metabolomic profiling of the algal extract, thirteen compounds were identified. Molecular docking of the identified compounds on the active site of the HMGB-1 protein revealed that 12,13-DiHome had the highest docking score of -7.130 kcal/mol. SPNP-gel showed higher wound closure potential and enhanced histopathological alterations as compared to standard (MEBO® ointment) and S. platensis gel in wounded Sprague-Dawley rats. Collectively, NPS promoted the wound healing process by enhancing the autophagy process (LC3B/Beclin-1) and the NRF-2/HO-1antioxidant pathway and halting the inflammatory (TNF-, NF-κB, TlR-4 and VEGF), apoptotic processes (AIF, Caspase-3), and the downregulation of HGMB-1 protein expression. The present study's findings suggest that the topical application of SPNP-gel possesses a potential therapeutic effect in excisional wound healing, chiefly by downregulating HGMB-1 protein expression.

Neoproterozoic origin and multiple transitions to macroscopic growth in green seaweeds.

The Neoproterozoic Era records the transition from a largely bacterial to a predominantly eukaryotic phototrophic world, creating the foundation for the complex benthic ecosystems that have sustained Metazoa from the Ediacaran Period onward. This study focuses on the evolutionary origins of green seaweeds, which play an important ecological role in the benthos of modern sunlit oceans and likely played a crucial part in the evolution of early animals by structuring benthic habitats and providing novel niches. By applying a phylogenomic approach, we resolve deep relationships of the core Chlorophyta (Ulvophyceae or green seaweeds, and freshwater or terrestrial Chlorophyceae and Trebouxiophyceae) and unveil a rapid radiation of Chlorophyceae and the principal lineages of the Ulvophyceae late in the Neoproterozoic Era. Our time-calibrated tree points to an origin and early diversification of green seaweeds in the late Tonian and Cryogenian periods, an interval marked by two global glaciations with strong consequent changes in the amount of available marine benthic habitat. We hypothesize that unicellular and simple multicellular ancestors of green seaweeds survived these extreme climate events in isolated refugia, and diversified in benthic environments that became increasingly available as ice retreated. An increased supply of nutrients and biotic interactions, such as grazing pressure, likely triggered the independent evolution of macroscopic growth via different strategies, including true multicellularity, and multiple types of giant-celled forms.

Shared up-regulation and contrasting down-regulation of gene expression distinguish desiccation-tolerant from intolerant green algae.

Among green plants, desiccation tolerance is common in seeds and spores but rare in leaves and other vegetative green tissues. Over the last two decades, genes have been identified whose expression is induced by desiccation in diverse, desiccation-tolerant (DT) taxa, including, e.g., late embryogenesis abundant proteins (LEA) and reactive oxygen species scavengers. This up-regulation is observed in DT resurrection plants, mosses, and green algae most closely related to these Embryophytes. Here we test whether this same suite of protective genes is up-regulated during desiccation in even more distantly related DT green algae, and, importantly, whether that up-regulation is unique to DT algae or also occurs in a desiccation-intolerant relative. We used three closely related aquatic and desert-derived green microalgae in the family Scenedesmaceae and capitalized on extraordinary desiccation tolerance in two of the species, contrasting with desiccation intolerance in the third. We found that during desiccation, all three species increased expression of common protective genes. The feature distinguishing gene expression in DT algae, however, was extensive down-regulation of gene expression associated with diverse metabolic processes during the desiccation time course, suggesting a switch from active growth to energy-saving metabolism. This widespread downshift did not occur in the desiccation-intolerant taxon. These results show that desiccation-induced up-regulation of expression of protective genes may be necessary but is not sufficient to confer desiccation tolerance. The data also suggest that desiccation tolerance may require induced protective mechanisms operating in concert with massive down-regulation of gene expression controlling numerous other aspects of metabolism.

Water oxidation by photosystem II is the primary source of electrons for sustained H2 photoproduction in nutrient-replete green algae.

The unicellular green alga Chlamydomonas reinhardtii is capable of photosynthetic H2 production. H2 evolution occurs under anaerobic conditions and is difficult to sustain due to 1) competition between [FeFe]-hydrogenase (H2ase), the key enzyme responsible for H2 metabolism in algae, and the Calvin-Benson-Bassham (CBB) cycle for photosynthetic reductants and 2) inactivation of H2ase by O2 coevolved in photosynthesis. Recently, we achieved sustainable H2 photoproduction by shifting algae from continuous illumination to a train of short (1 s) light pulses, interrupted by longer (9 s) dark periods. This illumination regime prevents activation of the CBB cycle and redirects photosynthetic electrons to H2ase. Employing membrane-inlet mass spectrometry and [Formula: see text], we now present clear evidence that efficient H2 photoproduction in pulse-illuminated algae depends primarily on direct water biophotolysis, where water oxidation at the donor side of photosystem II (PSII) provides electrons for the reduction of protons by H2ase downstream of photosystem I. This occurs exclusively in the absence of CO2 fixation, while with the activation of the CBB cycle by longer (8 s) light pulses the H2 photoproduction ceases and instead a slow overall H2 uptake is observed. We also demonstrate that the loss of PSII activity in DCMU-treated algae or in PSII-deficient mutant cells can be partly compensated for by the indirect (PSII-independent) H2 photoproduction pathway, but only for a short (<1 h) period. Thus, PSII activity is indispensable for a sustained process, where it is responsible for more than 92% of the final H2 yield.