Overexpression of the plastidial pseudo-protease AtFtsHi3 enhances drought tolerance while sustaining plant growth.

With climate change, droughts are expected to be more frequent and severe, severely impacting plant biomass and quality. Here, we show that overexpressing the Arabidopsis gene AtFtsHi3 (FtsHi3OE) enhances drought-tolerant phenotypes without compromising plant growth. AtFtsHi3 encodes a chloroplast envelope pseudo-protease; knock-down mutants (ftshi3-1) are found to be drought tolerant but exhibit stunted growth. Altered AtFtsHi3 expression therefore leads to drought tolerance, while only diminished expression of this gene leads to growth retardation. To understand the underlying mechanisms of the enhanced drought tolerance, we compared the proteomes of ftshi3-1 and pFtsHi3-FtsHi3OE (pFtsHi3-OE) to wild-type plants under well-watered and drought conditions. Drought-related processes like osmotic stress, water transport, and abscisic acid response were enriched in pFtsHi3-OE and ftshi3-1 mutants following their enhanced drought response compared to wild-type. The knock-down mutant ftshi3-1 showed an increased abundance of HSP90, HSP93, and TIC110 proteins, hinting at a potential downstream role of AtFtsHi3 in chloroplast pre-protein import. Mathematical modeling was performed to understand how variation in the transcript abundance of AtFtsHi3 can, on the one hand, lead to drought tolerance in both overexpression and knock-down lines, yet, on the other hand, affect plant growth so differently. The results led us to hypothesize that AtFtsHi3 may form complexes with at least two other protease subunits, either as homo- or heteromeric structures. Enriched amounts of AtFtsH7/9, AtFtsH11, AtFtsH12, and AtFtsHi4 in ftshi3-1 suggest a possible compensation mechanism for these proteases in the hexamer.

Nordic microalgae produce biostimulant for the germination of tomato and barley seeds.

Microalgal biomass may have biostimulating effects on plants and seeds due to its phytohormonal content, and harnessing this biostimulating effect could contribute to sustainable agriculture. Two Nordic strains of freshwater microalgae species Chlorella vulgaris and Scenedesmus obliquus were each cultivated in a photobioreactor receiving untreated municipal wastewater. The algal biomass and the supernatant after algal cultivation were tested on tomato and barley seeds for biostimulating effects. Intact algal cells, broken cells, or harvest supernatant were applied to the seeds, and germination time, percentage and germination index were evaluated. Seeds treated with C. vulgaris, in particular intact cells or supernatant, had up to 25 percentage units higher germination percentage after 2 days and an overall significantly faster germination time (germinated on average between 0.5 and 1 day sooner) than seeds treated with S. obliquus or the control (water). The germination index was higher in C. vulgaris treatments than in the control for both tomato and barley, and this was observed for both broken and intact cells as well as supernatant. The Nordic strain of C. vulgaris cultivated in municipal wastewater thus shows potential for use as biostimulant in agriculture, adding novel economic and sustainability benefits.

Biopolymer production from biomass produced by Nordic microalgae grown in wastewater.

Biomass from four different Nordic microalgal species, grown in BG-11 medium or synthetic wastewater (SWW), was explored as inexpensive carbohydrate-rich feedstock for polyhydroxybutyrate (PHB) production via microbial fermentation. Thermochemical pre-treatment (acid treatment followed by autoclavation) with 2% hydrochloric acid or 1% sulphuric acid (v/v) was used to maximize sugar yield prior to fermentation. Pre-treatment resulted in ∼5-fold higher sugar yield compared to the control. The sugar-rich hydrolysate was used as carbon source for the PHB-producing extremophilic bacterium Halomonas halophila. Maximal PHB production was achieved with hydrolysate of Chlorococcum sp. (MC-1) grown on BG-11 medium (0.27 ± 0.05 g PHB/ g DW), followed by hydrolysate derived from Desmodesmus sp. (RUC-2) grown on SWW (0.24 ± 0.05 g PHB/ g DW). Nordic microalgal biomass grown on wastewater therefore can be used as cheap feedstock for sustainable bioplastic production. This research highlights the potential of Nordic microalgae to develop a biobased economy.

Enhanced wastewater bioremediation by a sulfur-based copolymer as scaffold for microalgae immobilization (AlgaPol).

In recent years, there has been an increasing concern related to the contamination of aqueous ecosystems by heavy metals, highlighting the need to improve the current techniques for remediation. This work intends to address the problem of removing heavy metals from waterbodies by combining two complementary methodologies: adsorption to a copolymer synthesized by inverse vulcanization of sulfur and vegetable oils and phytoremediation by the microalga Chlorella sorokiniana to enhance the metal adsorption. After studying the tolerance and growth of Chlorella sorokiniana in the presence of the copolymer, the adsorption of highly concentrated Cd2+ (50 mg L-1) by the copolymer and microalgae on their own and the combined immobilized system (AlgaPol) was compared. Additionally, adsorption studies have been performed on mixtures of the heavy metals Cd2+ and Cu2+ at a concentration of 8 mg L-1 each. AlgaPol biofilm is able to remove these metals from the growth medium by more than 90%. The excellent metal adsorption capacity of this biofilm can be kinetically described by a pseudo-second-order model.

Exploring Nordic microalgae as a potential novel source of antioxidant and bioactive compounds.

Nordic microalgae are a group of photosynthetic organisms acclimated to growth at low temperature and in varying light conditions; the subarctic climate offers bright days with moderate temperatures during summer and cold and dark winter months. The robustness to these natural stress conditions makes the species interesting for large-scale cultivation in harsh environments and for the production of high-value compounds. The aim of this study was to explore the ability of nineteen species of Nordic microalgae to produce different bioactive compounds, such as carotenoids or polyphenols. The results showed that some of these strains are able to produce high amounts of carotenoids (over 12 mg·g-1 dry weight) and phenolic compounds (over 20 mg GAE·g-1 dry weight). Based on these profiles, six species were selected for cultivation under high light and cold stress (500 µmol·m-2·s-1 and 10 ˚C). The strains Chlorococcum sp. (MC1) and Scenedesmus sp. (B2-2) exhibited similar values of biomass productivity under standard or stress conditions, but produced higher concentrations of carotenoids (an increase of 40% and 25%, respectively), phenolic compounds (an increase of 40% and 30%, respectively), and showed higher antioxidant capacity (an increase of 15% and 20%, respectively) during stress. The results highlight the ability of these Nordic microalgae as outstanding producers of bioactive compounds, justifying their cultivation at large scale in Nordic environments.

Activity-based probes trap early active intermediates during metacaspase activation.

Metacaspases are essential cysteine proteases present in plants, fungi, and protists that are regulated by calcium binding and proteolytic maturation through mechanisms not yet understood. Here, we developed and validated activity-based probes for the three main metacaspase types, and used them to study calcium-mediated activation of metacaspases from their precursors in vitro. By combining substrate-inspired tetrapeptide probes containing an acyloxymethylketone (AOMK) reactive group, with purified representatives of type-I, type-II, and type-III metacaspases, we were able to demonstrate that labeling of mature metacaspases is strictly dependent on calcium. The probe with the highest affinity for all metacaspases also labels higher molecular weight proteoforms of all three metacaspases only in the presence of calcium, displaying the active, unprocessed metacaspase intermediates. Our data suggest that metacaspase activation proceeds through previously unknown active intermediates that are formed upon calcium binding, before precursor processing.

Detailed Characterization of the Cell Wall Structure and Composition of Nordic Green Microalgae.

Green microalgae are attractive to food, pharmaceutical, and biofuel industries due to the promising and diverse properties of their intracellular components. In current biotechnological applications, however, clear bottlenecks are the cell disruption and cell harvesting steps. Challenges in both of these processes are directly linked to the properties of the microalgal cell wall. The aim of this study was to explore the cell wall compositions and morphologies of four Nordic microalgal strains (Chlorella vulgaris (13-1), Scenedesmus sp. (B2-2), Haematococcus pluvialis, and Coelastrella sp. (3-4)) and their changes in relation to logarithmic and stationary growth phases. Transmission electron microscopy imaging enabled us to visualize the cell walls and to observe structural elements such as spines, microfibrillar hairs, or layers. Using cryogenic X-ray photoelectron spectroscopy, we quantified lipid, protein, and polysaccharide content of the outer surface of the microalgal cell wall in cultures. Fourier transform infrared spectroscopy highlighted changes between growth phases within the polysaccharide and protein fractions of the cell wall. Very prominent differences were observed in sugar and protein composition of the Scenedesmus sp. (B2-2) cell wall compared to the cell walls of the other three Nordic strains using trimethylsilyl derivatization.

Improving the content of high value compounds in Nordic Desmodesmus microalgal strains.

Nordic Desmodesmus microalgal strains (2-6) and (RUC-2) were exposed to abiotic stress (light and salt) to enhance lipids and carotenoids. The biomass output of both strains increased by more than 50% during light stress of 800 µmol m-2 s-1 compared to control light. The biomass of Desmodesmus sp. (2-6) contained most lipids (15% of dry weight) and total carotenoids (16.6 mg g-1) when grown at moderate light stress (400 µmol m-2 s-1), which further could be enhanced up to 2.5-fold by salinity stress. Desmodesmus sp. (RUC-2) exhibited maximal lipid (26.5%) and carotenoid (43.8 mg L-1) content at light intensities of 400 and 100 µmol m-2 s-1, respectively. Salinity stress stimulated lipid accumulation by 39%. Nordic Desmodesmus strains therefore are not only able to tolerate stress conditions, but their biomass considerably improves under stress. These strains have high potential to be used in algal bio-factories on low-cost medium like Baltic seawater.

Expression and Purification of the Type I Metacaspase from a Cryptophyte Guillardia theta , GtMCA-I.

Type I metacaspases are the most ubiquitous of the three metacaspase types and are present in representatives of prokaryotes, unicellular eukaryotes including yeasts, algae, and protozoa, as well as land plants. They are composed of two structural units: a catalytic so-called p20 domain with the His-Cys catalytic dyad and a regulatory p10 domain. Despite their structural homology to caspases, these proteases cleave their substrates after the positively charged amino acid residues at the P1 position, just like the metacaspases of type II and type III. We present a protocol for expression and purification of the only type I protease from a secondary endosymbiosis Guillardia theta , GtMCA-I by overexpression of its gene in BL21 (DE3) E. coli cells and one-day sequential purification using nickel-affinity, ion-exchange, and size-exclusion chromatography.

Expression and Purification of the Type II Metacaspase from a Unicellular Green Alga Chlamydomonas reinhardtii.

Type II metacaspases (MCAs) are proteases, belonging to the C14B MEROPS family. Like the MCAs of type I and type III, they preferentially cleave their substrates after the positively charged amino acid residues (Arg or Lys) at the P1 position. Type II MCAs from various higher plants have already been successfully overexpressed in E. coli mostly as His-tagged proteins and were shown to be proteolytically active after the purification. Here we present a protocol for expression and purification of the only type II MCA from the model green alga Chlamydomonas reinhardtii. The two-step purification, which consists of immobilized metal affinity chromatography using cobalt as ion followed by size-exclusion chromatography, can be performed in 1 day and yields 4 mg CrMCA-II protein per liter of overexpression culture.

A Review on Microbial Products and Their Perspective Application as Antimicrobial Agents.

Microorganisms including actinomycetes, archaea, bacteria, fungi, yeast, and microalgae are an auspicious source of vital bioactive compounds. In this review, the existing research regarding antimicrobial molecules from microorganisms is summarized. The potential antimicrobial compounds from actinomycetes, particularly Streptomyces spp.; archaea; fungi including endophytic, filamentous, and marine-derived fungi, mushroom; and microalgae are briefly described. Furthermore, this review briefly summarizes bacteriocins, halocins, sulfolobicin, etc., that target multiple-drug resistant pathogens and considers next-generation antibiotics. This review highlights the possibility of using microorganisms as an antimicrobial resource for biotechnological, nutraceutical, and pharmaceutical applications. However, more investigations are required to isolate, separate, purify, and characterize these bioactive compounds and transfer these primary drugs into clinically approved antibiotics.

The Plastid-Localized AtFtsHi3 Pseudo-Protease of Arabidopsis thaliana Has an Impact on Plant Growth and Drought Tolerance.

While drought severely affects plant growth and crop production, the molecular mechanisms of the drought response of plants remain unclear. In this study, we demonstrated for the first time the effect of the pseudo-protease AtFtsHi3 of Arabidopsis thaliana on overall plant growth and in drought tolerance. An AtFTSHi3 knock-down mutant [ftshi3-1(kd)] displayed a pale-green phenotype with lower photosynthetic efficiency and Darwinian fitness compared to wild type (Wt). An observed delay in seed germination of ftshi3-1(kd) was attributed to overaccumulation of abscisic acid (ABA); ftshi3-1(kd) seedlings showed partial sensitivity to exogenous ABA. Being exposed to similar severity of soil drying, ftshi3-1(kd) was drought-tolerant up to 20 days after the last irrigation, while wild type plants wilted after 12 days. Leaves of ftshi3-1(kd) contained reduced stomata size, density, and a smaller stomatic aperture. During drought stress, ftshi3-1(kd) showed lowered stomatal conductance, increased intrinsic water-use efficiency (WUEi), and slower stress acclimation. Expression levels of ABA-responsive genes were higher in leaves of ftshi3-1(kd) than Wt; DREB1A, but not DREB2A, was significantly upregulated during drought. However, although ftshi3-1(kd) displayed a drought-tolerant phenotype in aboveground tissue, the root-associated bacterial community responded to drought.

The FtsHi Enzymes of Arabidopsis thaliana: Pseudo-Proteases with an Important Function.

FtsH metalloproteases found in eubacteria, animals, and plants are well-known for their vital role in the maintenance and proteolysis of membrane proteins. Their location is restricted to organelles of endosymbiotic origin, the chloroplasts, and mitochondria. In the model organism Arabidopsis thaliana, there are 17 membrane-bound FtsH proteases containing an AAA+ (ATPase associated with various cellular activities) and a Zn2+ metalloprotease domain. However, in five of those, the zinc-binding motif HEXXH is either mutated (FtsHi1, 2, 4, 5) or completely missing (FtsHi3), rendering these enzymes presumably inactive in proteolysis. Still, homozygous null mutants of the pseudo-proteases FtsHi1, 2, 4, 5 are embryo-lethal. Homozygous ftshi3 or a weak point mutant in FTSHi1 are affected in overall plant growth and development. This review will focus on the findings concerning the FtsHi pseudo-proteases and their involvement in protein import, leading to consequences in embryogenesis, seed growth, chloroplast, and leaf development and oxidative stress management.

Wastewater treatment by microalgae.

The growth of the world's population increases the demand for fresh water, food, energy, and technology, which in turn leads to increasing amount of wastewater, produced both by domestic and industrial sources. These different wastewaters contain a wide variety of organic and inorganic compounds which can cause tremendous environmental problems if released untreated. Traditional treatment systems are usually expensive, energy demanding and are often still incapable of solving all challenges presented by the produced wastewaters. Microalgae are promising candidates for wastewater reclamation as they are capable of reducing the amount of nitrogen and phosphate as well as other toxic compounds including heavy metals or pharmaceuticals. Compared to the traditional systems, photosynthetic microalgae require less energy input since they use sunlight as their energy source, and at the same time lower the carbon footprint of the overall reclamation process. This mini-review focuses on recent advances in wastewater reclamation using microalgae. The most common microalgal strains used for this purpose are described as well as the challenges of using wastewater from different origins. We also describe the impact of climate with a particular focus on a Nordic climate.

The cell wall of green microalgae and its role in heavy metal removal.

Heavy metals in industrial wastewaters are posing a serious threat to the environment and to human health. Microalgae are increasingly being seen as potential solutions to this problem as they can remove pollutants through biosorption. This process offers certain advantages over other more traditional metal removal techniques as it is simple, inexpensive, eco-friendly, and can be performed over a wide range of experimental conditions. Biosorption is possible due to the unique and complex structure of the microalgal cell wall. The variety of functional groups on the surface of the cell wall (such as carboxyl or amino groups) can act as binding sites for the heavy metals, thus removing them from the environment. This review focuses on the cell wall composition and structure of the most commonly used microalgae in heavy metal removal and shows the role of their cell wall in the biosorption process. This review also aims to report the most commonly used models to predict the velocity of microalgal biosorption and the removal capacities.

Fate of active pharmaceutical ingredients in a northern high-rate algal pond fed with municipal wastewater.

Active pharmaceutical ingredients (APIs) are vital to human health and welfare, but following therapeutic use, they may pose a potential ecological risk if discharged into the environment. Today's conventional municipal wastewater treatment plants are not designed to remove APIs specifically, and various techniques, preferably cost-effective and environmentally friendly, are being developed and evaluated. Microalgae-based treatment of wastewater is a sustainable and low-cost approach to remove nutrients and emerging contaminants. In this study, a North Sweden high-rate algal pond (HRAP) using municipal untreated wastewater as medium, was investigated in terms of API distribution and fate. Three six-day batches were prepared during 18 days and a total of 36 APIs were quantified within the HRAP of which 14 were removed from the aqueous phase above 50% and seven removed above 90% of their initial concentrations. Twelve APIs of a hydrophobic nature were mostly associated with the algal biomass that was harvested at the end of each batch. HRAPs treatment successfully removed 69% of studied APIs (25 of 36 studied) in six day time. The distribution of various APIs between the aqueous phase and biomass suggested that several removal mechanisms may occur, such as hydrophobicity driven removal, passive biosorption and active bioaccumulation.

NordAqua, a Nordic Center of Excellence to develop an algae-based photosynthetic production platform.

NordAqua is a multidisciplinary Nordic Center of Excellence funded by NordForsk Bioeconomy program (2017-2022). The research center promotes Blue Bioeconomy and endeavours to reform the use of natural resources in a environmentally sustainable way. In this short communication, we summarize particular outcomes of the consortium. The key research progress of NordAqua includes (1) improving of photosynthetisis, (2) developing novel photosynthetic cell factories that function in a "solar-driven direct CO2 capture to target bioproducts" mode, (3) promoting the diversity of Nordic cyanobacteria and algae as an abundant and resilient alternative for less sustainable forest biomass and for innovative production of biochemicals, and (4) improving the bio-based wastewater purification and nutrient recycling technologies to provide new tools for integrative circular economy platforms.

Loss of Arabidopsis matrix metalloproteinase-5 affects root development and root bacterial communities during drought stress.

Matrix metalloproteinases (MMPs) are zinc-dependent endo-peptidases that in mammals are known to be involved in remodeling the extracellular matrix (ECM) in developmental and pathological processes. In this study, we report At5-MMP of Arabidopsis thaliana to be important for root development and root bacterial communities. At5-MMP is mainly localized in the root vasculature and lateral root, an At5-MMP T-DNA insertion mutant (mmp5 KO) showed reduced root growth and a lower number of root apexes, causing reduced water uptake from the soil. Subsequently, mmp5 KO is sensitive to drought stress. Inhibited auxin transport was accompanied with resistance to indole-3-acetic acid (IAA), 2, 4-dichlorophenoxyacetic acid (2, 4-D), and 1-naphthaleneacetic acid (NAA). The content of endogenous abscisic acid (ABA) was lower in roots of mmp5 KO than in wild type. Genes responsive to ABA as well as genes encoding enzymes of the proline biosynthesis were expressed to a lower extent in mmp5 KO than in wild type. Moreover, drought stress modulated root-associated bacterial communities of mmp5 KO: the number of Actinobacteria increased. Therefore, At5-MMP modulates auxin/ABA signaling rendering the plant sensitive to drought stress and recruiting differential root bacterial communities.

The Role of Pseudo-Orthocaspase (SyOC) of Synechocystis sp. PCC 6803 in Attenuating the Effect of Oxidative Stress.

Caspases are proteases, best known for their involvement in the execution of apoptosis-a subtype of programmed cell death, which occurs only in animals. These proteases are composed of two structural building blocks: a proteolytically active p20 domain and a regulatory p10 domain. Although structural homologs appear in representatives of all other organisms, their functional homology, i.e., cell death depending on their proteolytical activity, is still much disputed. Additionally, pseudo-caspases and pseudo-metacaspases, in which the catalytic histidine-cysteine dyad is substituted with non-proteolytic amino acid residues, were shown to be involved in cell death programs. Here, we present the involvement of a pseudo-orthocaspase (SyOC), a prokaryotic caspase-homolog lacking the p10 domain, in oxidative stress in the model cyanobacterium Synechocystis sp. PCC 6803. To study the in vivo impact of this pseudo-protease during oxidative stress its gene expression during exposure to H2O2 was monitored by RT-qPCR. Furthermore, a knock-out mutant lacking the pseudo-orthocaspase gene was designed, and its survival and growth rates were compared to wild type cells as well as its proteome. Deletion of SyOC led to cells with a higher tolerance toward oxidative stress, suggesting that this protein may be involved in a pro-death pathway.