A candidate transporter allowing symbiotic dinoflagellates to feed their coral hosts.

The symbiotic partnership between corals and dinoflagellate algae is crucial to coral reefs. Corals provide their algal symbionts with shelter, carbon dioxide and nitrogen. In exchange, the symbiotic algae supply their animal hosts with fixed carbon in the form of glucose. But how glucose is transferred from the algal symbiont to the animal host is unknown. We reasoned that a transporter resident in the dinoflagellate cell membrane would facilitate outward transfer of glucose to the surrounding host animal tissue. We identified a candidate transporter in the cnidarian symbiont dinoflagellate Breviolum minutum that belongs to the ubiquitous family of facilitative sugar uniporters known as SWEETs (sugars will eventually be exported transporters). Previous gene expression analyses had shown that BmSWEET1 is upregulated when the algae are living symbiotically in a cnidarian host by comparison to the free-living state [1, 2]. We used immunofluorescence microscopy to localise BmSWEET1 in the dinoflagellate cell membrane. Substrate preference assays in a yeast surrogate transport system showed that BmSWEET1 transports glucose. Quantitative microscopy showed that symbiotic B. minutum cells have significantly more BmSWEET1 protein than free-living cells of the same strain, consistent with export during symbiosis but not during the free-living, planktonic phase. Thus, BmSWEET1 is in the right place, at the right time, and has the right substrate to be the transporter with which symbiotic dinoflagellate algae feed their animal hosts to power coral reefs.

The Influence of a Cryptochrome on the Gene Expression Profile in the Diatom Phaeodactylum tricornutum under Blue Light and in Darkness.

Diatoms, albeit being only distantly related with higher plants, harbor a plant-like cryptochrome (CryP) that was proposed to act as a photoreceptor required for the regulation of some photosynthetic proteins. Plant cryptochromes are involved in the regulation of developmental processes relevant only to multicellular organisms. Their role in the unicellular diatoms to date is mostly enigmatic. To elucidate the function of this plant-like cryptochrome in a unicellular species, we examined the role of CryP in the regulation of transcription in the diatom Phaeodactylum tricornutum by comparative RNA-seq of wild type and CryP knock-down mutants, under prolonged darkness and one hour after onset of blue light. In total, mRNAs of 12,298 genes were identified and more than 70% of the genes could be sorted into functional bins. CryP influenced groups of transcripts in three different ways: some transcripts displayed altered expression under blue light only, others independent of the light condition, and, surprisingly, some were influenced by CryP only in darkness. Genes regulated in any condition were distributed over almost all functional categories. CryP exerted an influence on two other photoreceptors: the genes encoding phytochrome and CPF1, another cryptochrome, which were down-regulated by CryP independent of the light condition. However, the regulatory responses of the affected photoreceptors on transcriptional output were independent. The influence of CryP on the expression of other photoreceptors hints to the existence of a regulatory signaling network in diatoms that includes several cryptochromes and phytochrome, whereby CryP acts as a regulator of transcript abundance under light as well as in darkness.

Transcriptional response of the extremophile red alga Cyanidioschyzon merolae to changes in CO2 concentrations.

Cyanidioschyzon merolae (C. merolae) is an acidophilic red alga growing in a naturally low carbon dioxide (CO2) environment. Although it uses a ribulose 1,5-bisphosphate carboxylase/oxygenase with high affinity for CO2, the survival of C. merolae relies on functional photorespiratory metabolism. In this study, we quantified the transcriptomic response of C. merolae to changes in CO2 conditions. We found distinct changes upon shifts between CO2 conditions, such as a concerted up-regulation of photorespiratory genes and responses to carbon starvation. We used the transcriptome data set to explore a hypothetical CO2 concentrating mechanism in C. merolae, based on the assumption that photorespiratory genes and possible candidate genes involved in a CO2 concentrating mechanism are co-expressed. A putative bicarbonate transport protein and two α-carbonic anhydrases were identified, which showed enhanced transcript levels under reduced CO2 conditions. Genes encoding enzymes of a PEPCK-type C4 pathway were co-regulated with the photorespiratory gene cluster. We propose a model of a hypothetical low CO2 compensation mechanism in C. merolae integrating these low CO2-inducible components.

The Synechocystis Manganese Exporter Mnx Is Essential for Manganese Homeostasis in Cyanobacteria.

The essential micronutrient manganese (Mn) functions as redox-active cofactor in active sites of enzymes and, thus, is involved in various physiological reactions. Moreover, in oxygenic photosynthetic organisms, Mn is of special importance, since it is central to the oxygen-evolving complex in photosystem II. Although Mn is an essential micronutrient, increased amounts are detrimental to the organism; thus, only a small window exists for beneficial concentrations. Accordingly, Mn homeostasis must be carefully maintained. In contrast to the well-studied uptake mechanisms in cyanobacteria, it is largely unknown how Mn is distributed to the different compartments inside the cell. We identified a protein with so far unknown function as a hypothetical Mn transporter in the cyanobacterial model strain Synechocystis sp. PCC 6803 and named this protein Mnx for Mn exporter. The knockout mutant Δmnx showed increased sensitivity toward externally supplied Mn and Mn toxicity symptoms, which could be linked to intracellular Mn accumulation. 54Mn chase experiments demonstrated that the mutant was not able to release Mn from the internal pool. Microscopic analysis of a Mnx::yellow fluorescent protein fusion showed that the protein resides in the thylakoid membrane. Heterologous expression of mnx suppressed the Mn-sensitive phenotype of the Saccharomyces cerevisiae mutant Δpmr1 Our results indicate that Mnx functions as a thylakoid Mn transporter and is a key player in maintaining Mn homeostasis in Synechocystis sp. PCC 6803. We propose that Mn export from the cytoplasm into the thylakoid lumen is crucial to prevent toxic cytoplasmic Mn accumulation and to ensure Mn provision to photosystem II.

Photosynthesis in C3-C4 intermediate Moricandia species.

Evolution of C4 photosynthesis is not distributed evenly in the plant kingdom. Particularly interesting is the situation in the Brassicaceae, because the family contains no C4 species, but several C3-C4 intermediates, mainly in the genus Moricandia Investigation of leaf anatomy, gas exchange parameters, the metabolome, and the transcriptome of two C3-C4 intermediate Moricandia species, M. arvensis and M. suffruticosa, and their close C3 relative M. moricandioides enabled us to unravel the specific C3-C4 characteristics in these Moricandia lines. Reduced CO2 compensation points in these lines were accompanied by anatomical adjustments, such as centripetal concentration of organelles in the bundle sheath, and metabolic adjustments, such as the balancing of C and N metabolism between mesophyll and bundle sheath cells by multiple pathways. Evolution from C3 to C3-C4 intermediacy was probably facilitated first by loss of one copy of the glycine decarboxylase P-protein, followed by dominant activity of a bundle sheath-specific element in its promoter. In contrast to recent models, installation of the C3-C4 pathway was not accompanied by enhanced activity of the C4 cycle. Our results indicate that metabolic limitations connected to N metabolism or anatomical limitations connected to vein density could have constrained evolution of C4 in Moricandia.

Overexpression of the triose phosphate translocator (TPT) complements the abnormal metabolism and development of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase mutants.

The presence of two glycolytic pathways working in parallel in plastids and cytosol has complicated the understanding of this essential process in plant cells, especially the integration of the plastidial pathway into the metabolism of heterotrophic and autotrophic organs. It is assumed that this integration is achieved by transport systems, which exchange glycolytic intermediates across plastidial membranes. However, it is unknown whether plastidial and cytosolic pools of 3-phosphoglycerate (3-PGA) can equilibrate in non-photosynthetic tissues. To resolve this question, we employed Arabidopsis mutants of the plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) that express the triose phosphate translocator (TPT) under the control of the 35S (35S:TPT) or the native GAPCp1 (GAPCp1:TPT) promoters. TPT expression under the control of both promoters complemented the vegetative developmental defects and metabolic disorders of the GAPCp double mutants (gapcp1gapcp2). However, as the 35S is poorly expressed in the tapetum, full vegetative and reproductive complementation of gapcp1gapcp2 was achieved only by transforming this mutant with the GAPCp1:TPT construct. Our results indicate that the main function of GAPCp is to supply 3-PGA for anabolic pathways in plastids of heterotrophic cells and suggest that the plastidial glycolysis may contribute to fatty acid biosynthesis in seeds. They also suggest a 3-PGA deficiency in the plastids of gapcp1gapcp2, and that 3-PGA pools between cytosol and plastid do not equilibrate in heterotrophic cells.

The Peroxisomal NAD Carrier from Arabidopsis Imports NAD in Exchange with AMP.

Cofactors such as NAD, AMP, and Coenzyme A (CoA) are essential for a diverse set of reactions and pathways in the cell. Specific carrier proteins are required to distribute these cofactors to different cell compartments, including peroxisomes. We previously identified a peroxisomal transport protein in Arabidopsis (Arabidopsis thaliana) called the peroxisomal NAD carrier (PXN). When assayed in vitro, this carrier exhibits versatile transport functions, e.g. catalyzing the import of NAD or CoA, the exchange of NAD/NADH, and the export of CoA. These observations raise the question about the physiological function of PXN in plants. Here, we used Saccharomyces cerevisiae to address this question. First, we confirmed that PXN, when expressed in yeast, is active and targeted to yeast peroxisomes. Secondl, detailed uptake analyses revealed that the CoA transport function of PXN can be excluded under physiological conditions due to its low affinity for this substrate. Third, we expressed PXN in diverse mutant yeast strains and investigated the suppression of the mutant phenotypes. These studies provided strong evidences that PXN was not able to function as a CoA transporter or a redox shuttle by mediating a NAD/NADH exchange, but instead catalyzed the import of NAD into peroxisomes against AMP in intact yeast cells.

The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis.

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn(2+) and Ca(2+) homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn(2+) and Ca(2+) ions were differently sequestered in pam71, with Ca(2+) enriched in pam71 thylakoids relative to the wild type. The changes in Ca(2+) homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn(2+), but not Ca(2+) Furthermore, PAM71 suppressed the Mn(2+)-sensitive phenotype of the yeast mutant Δpmr1 Therefore, PAM71 presumably functions in Mn(2+) uptake into thylakoids to ensure optimal PSII performance.

Azolla domestication towards a biobased economy?

Due to its phenomenal growth requiring neither nitrogen fertilizer nor arable land and its biomass composition, the mosquito fern Azolla is a candidate crop to yield food, fuels and chemicals sustainably. To advance Azolla domestication, we research its dissemination, storage and transcriptome. Methods for dissemination, cross-fertilization and cryopreservation of the symbiosis Azolla filiculoides-Nostoc azollae are tested based on the fern spores. To study molecular processes in Azolla including spore induction, a database of 37 649 unigenes from RNAseq of microsporocarps, megasporocarps and sporophytes was assembled, then validated. Spores obtained year-round germinated in vitro within 26 d. In vitro fertilization rates reached 25%. Cryopreservation permitted storage for at least 7 months. The unigene database entirely covered central metabolism and to a large degree covered cellular processes and regulatory networks. Analysis of genes engaged in transition to sexual reproduction revealed a FLOWERING LOCUS T-like protein in ferns with special features induced in sporulating Azolla fronds. Although domestication of a fern-cyanobacteria symbiosis may seem a daunting task, we conclude that the time is ripe and that results generated will serve to more widely access biochemicals in fern biomass for a biobased economy.

Abnormal glycosphingolipid mannosylation triggers salicylic acid-mediated responses in Arabidopsis.

The Arabidopsis thaliana protein GOLGI-LOCALIZED NUCLEOTIDE SUGAR TRANSPORTER (GONST1) has been previously identified as a GDP-d-mannose transporter. It has been hypothesized that GONST1 provides precursors for the synthesis of cell wall polysaccharides, such as glucomannan. Here, we show that in vitro GONST1 can transport all four plant GDP-sugars. However, gonst1 mutants have no reduction in glucomannan quantity and show no detectable alterations in other cell wall polysaccharides. By contrast, we show that a class of glycosylated sphingolipids (glycosylinositol phosphoceramides [GIPCs]) contains Man and that this mannosylation is affected in gonst1. GONST1 therefore is a Golgi GDP-sugar transporter that specifically supplies GDP-Man to the Golgi lumen for GIPC synthesis. gonst1 plants have a dwarfed phenotype and a constitutive hypersensitive response with elevated salicylic acid levels. This suggests an unexpected role for GIPC sugar decorations in sphingolipid function and plant defense signaling. Additionally, we discuss these data in the context of substrate channeling within the Golgi.