Pumping iron: A multi-omics analysis of two extremophilic algae reveals iron economy management.

Marine algae are responsible for half of the world's primary productivity, but this critical carbon sink is often constrained by insufficient iron. One species of marine algae, Dunaliella tertiolecta, is remarkable for its ability to maintain photosynthesis and thrive in low-iron environments. A related species, Dunaliella salina Bardawil, shares this attribute but is an extremophile found in hypersaline environments. To elucidate how algae manage their iron requirements, we produced high-quality genome assemblies and transcriptomes for both species to serve as a foundation for a comparative multiomics analysis. We identified a host of iron-uptake proteins in both species, including a massive expansion of transferrins and a unique family of siderophore-iron-uptake proteins. Complementing these multiple iron-uptake routes, ferredoxin functions as a large iron reservoir that can be released by induction of flavodoxin. Proteomic analysis revealed reduced investment in the photosynthetic apparatus coupled with remodeling of antenna proteins by dramatic iron-deficiency induction of TIDI1, which is closely related but identifiably distinct from the chlorophyll binding protein, LHCA3. These combinatorial iron scavenging and sparing strategies make Dunaliella unique among photosynthetic organisms.

Widespread polycistronic gene expression in green algae.

Polycistronic gene expression, common in prokaryotes, was thought to be extremely rare in eukaryotes. The development of long-read sequencing of full-length transcript isomers (Iso-Seq) has facilitated a reexamination of that dogma. Using Iso-Seq, we discovered hundreds of examples of polycistronic expression of nuclear genes in two divergent species of green algae: Chlamydomonas reinhardtii and Chromochloris zofingiensis Here, we employ a range of independent approaches to validate that multiple proteins are translated from a common transcript for hundreds of loci. A chromatin immunoprecipitation analysis using trimethylation of lysine 4 on histone H3 marks confirmed that transcription begins exclusively at the upstream gene. Quantification of polyadenylated [poly(A)] tails and poly(A) signal sequences confirmed that transcription ends exclusively after the downstream gene. Coexpression analysis found nearly perfect correlation for open reading frames (ORFs) within polycistronic loci, consistent with expression in a shared transcript. For many polycistronic loci, terminal peptides from both ORFs were identified from proteomics datasets, consistent with independent translation. Synthetic polycistronic gene pairs were transcribed and translated in vitro to recapitulate the production of two distinct proteins from a common transcript. The relative abundance of these two proteins can be modified by altering the Kozak-like sequence of the upstream gene. Replacement of the ORFs with selectable markers or reporters allows production of such heterologous proteins, speaking to utility in synthetic biology approaches. Conservation of a significant number of polycistronic gene pairs between C. reinhardtii, C. zofingiensis, and five other species suggests that this mechanism may be evolutionarily ancient and biologically important in the green algal lineage.

A hypothesis about the origin of carotenoid lipid droplets in the green algae Dunaliella and Haematococcus.

MAIN CONCLUSION: Hypercarotenogenesis in green algae evolved by mutation of PSY that increased its transcription at high light, disintegration of the eyespot in Dunaliella and acquisition of the capacity to export carotenoids from chloroplasts in Haematococcus. Carotenoids (Car) are lipid-soluble pigments synthesized in plants, algae, bacteria and fungi. Car have strong antioxidative properties and as such are utilized to reduce the danger of different diseases in humans. Two green microalgae are utilized as rich natural sources for Car: Dunaliella salina/bardawil accumulates 10% (w/w) ß-carotene (ßC), which is also pro-vitamin A, and Haematococcus pluvialis accumulates 4% (w/w) astaxanthin (Ast), the strongest antioxidant among Car. D. bardawil accumulates ßC in plastoglobules within the chloroplast, whereas H. pluvialis deposits Ast in cytoplasmic lipid droplets (CLD). In this review we compare the hypercarotenogenic responses (HCR) in Dunaliella and in Haematococcus and try to outline hypothetical evolutionary pathways for its origin. We propose that a mutation in phytoene synthetase that increased its transcription level in response to high light stress had a pivotal role in the evolution of the HCR. Proteomic analyses indicated that in D. bardawil/salina the HCR evolved from dissociation and amplification of eyespot lipid globules. The more robust HCR in algae that accumulate carotenoids in CLD, such as H. pluvialis, required also acquisition of the capacity to export ßC out of the chloroplast and its enzymatic conversion into Ast.

Novel 9-cis/all-trans ß-carotene isomerases from plastidic oil bodies in Dunaliella bardawil catalyze the conversion of all-trans to 9-cis ß-carotene.

KEY MESSAGE: We identified and demonstrated the function of 9-cis/all-trans ß-carotene isomerases in plastidic globules of Dunaliella bardawil, the species accumulating the highest levels of 9-cis ß-carotene that is essential for humans. The halotolerant alga Dunaliella bardawil is unique in that it accumulates under light stress high levels of ß-carotene in plastidic lipid globules. The pigment is composed of two major isomers: all-trans ß-carotene, the common natural form of this pigment, and 9-cis ß-carotene. The biosynthetic pathway of ß-carotene is known, but it is not clear how the 9-cis isomer is formed. We identified in plastidic lipid globules that were isolated from D. bardawil two proteins with high sequence homology to the D27 protein-a 9-cis/all-trans ß-carotene isomerase from rice (Alder et al. Science 335:1348-1351, 2012). The proteins are enriched in the oil globules by 6- to 17-fold compared to chloroplast proteins. The expression of the corresponding genes, 9-cis-ßC-iso1 and 9-cis-ßC-iso2, is enhanced under light stress. The synthetic proteins catalyze in vitro conversion of all-trans to 9-cis ß-carotene. Expression of the 9-cis-ßC-iso1 or of 9-cis-ßC-iso2 genes in an E. coli mutant line that harbors ß-carotene biosynthesis genes enhanced the conversion of all-trans into 9-cis ß-carotene. These results suggest that 9-cis-ßC-ISO1 and 9-cis-ßC-ISO2 proteins are responsible for the formation of 9-cis ß-carotene in D. bardawil under stress conditions.

Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome.

Efficient breakdown of lignocellulose polymers into simple molecules is a key technological bottleneck limiting the production of plant-derived biofuels and chemicals. In nature, plant biomass degradation is achieved by the action of a wide range of microbial enzymes. In aerobic microorganisms, these enzymes are secreted as discrete elements in contrast to certain anaerobic bacteria, where they are assembled into large multienzyme complexes termed cellulosomes. These complexes allow for very efficient hydrolysis of cellulose and hemicellulose due to the spatial proximity of synergistically acting enzymes and to the limited diffusion of the enzymes and their products. Recently, designer cellulosomes have been developed to incorporate foreign enzymatic activities in cellulosomes so as to enhance lignocellulose hydrolysis further. In this study, we complemented a cellulosome active on cellulose and hemicellulose by addition of an enzyme active on lignin. To do so, we designed a dockerin-fused variant of a recently characterized laccase from the aerobic bacterium Thermobifida fusca The resultant chimera exhibited activity levels similar to the wild-type enzyme and properly integrated into the designer cellulosome. The resulting complex yielded a twofold increase in the amount of reducing sugars released from wheat straw compared with the same system lacking the laccase. The unorthodox use of aerobic enzymes in designer cellulosome machinery effects simultaneous degradation of the three major components of the plant cell wall (cellulose, hemicellulose, and lignin), paving the way for more efficient lignocellulose conversion into soluble sugars en route to alternative fuels production.

Proteome analysis of cytoplasmatic and plastidic ß-carotene lipid droplets in Dunaliella bardawil.

The halotolerant green alga Dunaliella bardawil is unique in that it accumulates under stress two types of lipid droplets: cytoplasmatic lipid droplets (CLD) and ß-carotene-rich (ßC) plastoglobuli. Recently, we isolated and analyzed the lipid and pigment compositions of these lipid droplets. Here, we describe their proteome analysis. A contamination filter and an enrichment filter were utilized to define core proteins. A proteome database of Dunaliella salina/D. bardawil was constructed to aid the identification of lipid droplet proteins. A total of 124 and 42 core proteins were identified in ßC-plastoglobuli and CLD, respectively, with only eight common proteins. Dunaliella spp. CLD resemble cytoplasmic droplets from Chlamydomonas reinhardtii and contain major lipid droplet-associated protein and enzymes involved in lipid and sterol metabolism. The ßC-plastoglobuli proteome resembles the C. reinhardtii eyespot and Arabidopsis (Arabidopsis thaliana) plastoglobule proteomes and contains carotene-globule-associated protein, plastid-lipid-associated protein-fibrillins, SOUL heme-binding proteins, phytyl ester synthases, ß-carotene biosynthesis enzymes, and proteins involved in membrane remodeling/lipid droplet biogenesis: VESICLE-INDUCING PLASTID PROTEIN1, synaptotagmin, and the eyespot assembly proteins EYE3 and SOUL3. Based on these and previous results, we propose models for the biogenesis of ßC-plastoglobuli and the biosynthesis of ß-carotene within ßC-plastoglobuli and hypothesize that ßC-plastoglobuli evolved from eyespot lipid droplets.

Origin of ß-carotene-rich plastoglobuli in Dunaliella bardawil.

The halotolerant microalgae Dunaliella bardawil accumulates under nitrogen deprivation two types of lipid droplets: plastoglobuli rich in ß-carotene (ßC-plastoglobuli) and cytoplasmatic lipid droplets (CLDs). We describe the isolation, composition, and origin of these lipid droplets. Plastoglobuli contain ß-carotene, phytoene, and galactolipids missing in CLDs. The two preparations contain different lipid-associated proteins: major lipid droplet protein in CLD and the Prorich carotene globule protein in ßC-plastoglobuli. The compositions of triglyceride (TAG) molecular species, total fatty acids, and sn-1+3 and sn-2 positions in the two lipid pools are similar, except for a small increase in palmitic acid in plastoglobuli, suggesting a common origin. The formation of CLD TAG precedes that of ßC-plastoglobuli, reaching a maximum after 48 h of nitrogen deprivation and then decreasing. Palmitic acid incorporation kinetics indicated that, at early stages of nitrogen deprivation, CLD TAG is synthesized mostly from newly formed fatty acids, whereas in ßC-plastoglobuli, a large part of TAG is produced from fatty acids of preformed membrane lipids. Electron microscopic analyses revealed that CLDs adhere to chloroplast envelope membranes concomitant with appearance of small ßC-plastoglobuli within the chloroplast. Based on these results, we propose that CLDs in D. bardawil are produced in the endoplasmatic reticulum, whereas ßC-plastoglobuli are made, in part, from hydrolysis of chloroplast membrane lipids and in part, by a continual transfer of TAG or fatty acids derived from CLD.

Characterization of major lipid droplet proteins from Dunaliella.

Many green algal species can accumulate large amounts of triacylglycerides (TAG) under nutrient deprivation, making them a potential source for production of biodiesel. TAG are organized in cytoplasmic lipid bodies, which contain a major lipid droplet protein termed MLDP. Green algae MLDP differ in sequence from plant oleosins and from animal perilipins, and their structure and function are not clear. In this study, we describe the isolation of MLDP from three species of the extreme halotolerant green algae Dunaliella. Sequence alignment with other green algae MLDP proteins identified a conserved 4-proline domain that may be considered as a signature domain of Volvocales green algae MLDP. Gold immunolabeling localized MLDP at the surface of lipid droplets in D. salina. The induction of MLDP by nitrogen deprivation is kinetically correlated with TAG accumulation, and inhibition of TAG biosynthesis impairs MLDP accumulation suggesting that MLDP induction is co-regulated with TAG accumulation. These results can lead to a better understanding of the structure and function of Volvocales green algae MLDP proteins.