Using Publicly Available RNA-seq Data for Expression Analysis of Genes of Interest.

RNA-seq data in publicly available repositories enable the efficient reanalysis of transcript abundances in existing experiments. Graphical user interfaces usually only allow the visual inspection of a single gene and of predefined experiments. Here, we describe how experiments are selected from the Sequence Read Archive or the European Nucleotide Archive, how data is efficiently mapped onto a reference transcriptome, and how global transcript abundances and patterns are inspected. We exemplarily apply this analysis pipeline to study the expression of photorespiration-related genes in photosynthetic organisms, such as cyanobacteria, and to identify conditions under which photorespiratory transcript abundances are enhanced.

Four plus one: vacuoles serve in photorespiration.

Photorespiration is inevitable for oxygenic photosynthesis. It has fascinated researchers over decades because of its multicompartmental organization. Recently, Lin and Tsay identified a vacuole glycerate transporter contributing to photorespiratory metabolism under short-term nitrogen depletion. This key finding adds a fifth interacting subcellular compartment and extends the photorespiratory metabolic repair module.

PYRIDOX(AM)INE 5'-PHOSPHATE OXIDASE3 of Arabidopsis thaliana maintains carbon/nitrogen balance in distinct environmental conditions.

The identification of factors that regulate C/N utilization in plants can make a substantial contribution to optimization of plant health. Here, we explored the contribution of pyridox(am)ine 5'-phosphate oxidase3 (PDX3), which regulates vitamin B6 homeostasis, in Arabidopsis (Arabidopsis thaliana). Firstly, N fertilization regimes showed that ammonium application rescues the leaf morphological phenotype of pdx3 mutant lines but masks the metabolite perturbance resulting from impairment in utilizing soil nitrate as a source of N. Without fertilization, pdx3 lines suffered a C/N imbalance and accumulated nitrogenous compounds. Surprisingly, exploration of photorespiration as a source of endogenous N driving this metabolic imbalance, by incubation under high CO2, further exacerbated the pdx3 growth phenotype. Interestingly, the amino acid serine, critical for growth and N management, alleviated the growth phenotype of pdx3 plants under high CO2, likely due to the requirement of pyridoxal 5'-phosphate for the phosphorylated pathway of serine biosynthesis under this condition. Triggering of thermomorphogenesis by growth of plants at 28 °C (instead of 22 °C) did not appear to require PDX3 function, and we observed that the consequent drive toward C metabolism counters the C/N imbalance in pdx3. Further, pdx3 lines suffered a salicylic acid-induced defense response, probing of which unraveled that it is a protective strategy mediated by nonexpressor of pathogenesis related1 (NPR1) and improves fitness. Overall, the study demonstrates the importance of vitamin B6 homeostasis as managed by the salvage pathway enzyme PDX3 to growth in diverse environments with varying nutrient availability and insight into how plants reprogram their metabolism under such conditions.

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 Full Genome Sequences of Pseudomonas sp. Strain MM221 and Pseudoarthrobacter sp. Strain MM222, Isolated from a Meadow in Bielefeld, Germany.

The bacterial strains Pseudomonas sp. strain MM221 and Pseudoarthrobacter sp. strain MM222 were isolated from a sandy soil sample. Here, we report on their complete genome sequences, including a circular plasmid for MM221, which were assembled after sequencing with an Oxford Nanopore Technologies flow cell.

Complete Genome Sequences of Pseudomonas sp. Strains MM223 and MM227 and Rheinheimera sp. Strain MM224, Isolated from a Pond Edge in Bielefeld, Germany.

Pseudomonas sp. strain MM223, Pseudomonas sp. strain MM227, and Rheinheimera sp. strain MM224 were isolated from a muddy soil sample from the edge of a pond. Here, we present whole-genome sequences and phylogenetic classifications for all three bacterial isolates.

Metabolite Profiling in Arabidopsisthaliana with Moderately Impaired Photorespiration Reveals Novel Metabolic Links and Compensatory Mechanisms of Photorespiration.

Photorespiration is an integral component of plant primary metabolism. Accordingly, it has been often observed that impairing the photorespiratory flux negatively impacts other cellular processes. In this study, the metabolic acclimation of the Arabidopsisthaliana wild type was compared with the hydroxypyruvate reductase 1 (HPR1; hpr1) mutant, displaying only a moderately reduced photorespiratory flux. Plants were analyzed during development and under varying photoperiods with a combination of non-targeted and targeted metabolome analysis, as well as 13C- and 14C-labeling approaches. The results showed that HPR1 deficiency is more critical for photorespiration during the vegetative compared to the regenerative growth phase. A shorter photoperiod seems to slowdown the photorespiratory metabolite conversion mostly at the glycerate kinase and glycine decarboxylase steps compared to long days. It is demonstrated that even a moderate impairment of photorespiration severely reduces the leaf-carbohydrate status and impacts on sulfur metabolism. Isotope labeling approaches revealed an increased CO2 release from hpr1 leaves, most likely occurring from enhanced non-enzymatic 3-hydroxypyruvate decarboxylation and a higher flux from serine towards ethanolamine through serine decarboxylase. Collectively, the study provides evidence that the moderate hpr1 mutant is an excellent tool to unravel the underlying mechanisms governing the regulation of metabolic linkages of photorespiration with plant primary metabolism.

Transport Proteins Enabling Plant Photorespiratory Metabolism.

Photorespiration (PR) is a metabolic repair pathway that acts in oxygenic photosynthetic organisms to degrade a toxic product of oxygen fixation generated by the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase. Within the metabolic pathway, energy is consumed and carbon dioxide released. Consequently, PR is seen as a wasteful process making it a promising target for engineering to enhance plant productivity. Transport and channel proteins connect the organelles accomplishing the PR pathway-chloroplast, peroxisome, and mitochondrion-and thus enable efficient flux of PR metabolites. Although the pathway and the enzymes catalyzing the biochemical reactions have been the focus of research for the last several decades, the knowledge about transport proteins involved in PR is still limited. This review presents a timely state of knowledge with regard to metabolite channeling in PR and the participating proteins. The significance of transporters for implementation of synthetic bypasses to PR is highlighted. As an excursion, the physiological contribution of transport proteins that are involved in C4 metabolism is discussed.

The Aureochrome Photoreceptor PtAUREO1a Is a Highly Effective Blue Light Switch in Diatoms.

Aureochromes represent a unique type of blue light photoreceptors that possess a blue light sensing flavin-binding LOV-domain and a DNA-binding bZIP domain, thus being light-driven transcription factors. The diatom Phaeodactylum tricornutum, a member of the essential marine primary producers, possesses four aureochromes (PtAUREO1a, 1b, 1c, 2). Here we show a dramatic change in the global gene expression pattern of P. tricornutum wild-type cells after a shift from red to blue light. About 75% of the genes show significantly changed transcript levels already after 10 and 60 min of blue light exposure, which includes genes of major transcription factors as well as other photoreceptors. Very surprisingly, this light-induced regulation of gene expression is almost completely inhibited in independent PtAureo1a knockout lines. Such a massive and fast transcriptional change depending on one single photoreceptor is so far unprecedented. We conclude that PtAUREO1a plays a key role in diatoms upon blue light exposure.

Chloroplast Transition Metal Regulation for Efficient Photosynthesis.

Plants require sunlight, water, CO2, and essential nutrients to drive photosynthesis and fulfill their life cycle. The photosynthetic apparatus resides in chloroplasts and fundamentally relies on transition metals as catalysts and cofactors. Accordingly, chloroplasts are particularly rich in iron (Fe), manganese (Mn), and copper (Cu). Owing to their redox properties, those metals need to be carefully balanced within the cell. However, the regulation of transition metal homeostasis in chloroplasts is poorly understood. With the availability of the arabidopsis genome information and membrane protein databases, a wider catalogue for searching chloroplast metal transporters has considerably advanced the study of transition metal regulation. This review provides an updated overview of the chloroplast transition metal requirements and the transporters involved for efficient photosynthesis in higher plants.

Manganese Homeostasis in Cyanobacteria.

Manganese (Mn) is essential for life on earth. As a catalyst of the water oxidation reaction within photosystem II, the trace metal is responsible for the evolution of virtually all oxygen in the earth's atmosphere. Mn acts furthermore as an activator or cofactor of numerous enzymes involved in reactive oxygen species scavenging or central and secondary metabolism. While the sufficient supply of oxygenic photosynthetic organisms with Mn is obvious for maintaining photosynthetic activity, the avoidance of cellular Mn overload is also critical. In this review, current knowledge about the Mn homeostasis network in the model cyanobacterium Synechocystis sp. PCC 6803 is presented, including transporters and regulators.

Mechanistic understanding of photorespiration paves the way to a new green revolution.

Photorespiration is frequently considered a wasteful and inefficient process. However, mutant analysis demonstrated that photorespiration is essential for recycling of 2-phosphoglycolate in C3 and C4 land plants, in algae, and even in cyanobacteria operating carboxysome-based carbon (C) concentrating mechanisms. Photorespiration links photosynthetic C assimilation with other metabolic processes, such as nitrogen and sulfur assimilation, as well as C1 metabolism, and it may contribute to balancing the redox poise between chloroplasts, peroxisomes, mitochondria and cytoplasm. The high degree of metabolic interdependencies and the pleiotropic phenotypes of photorespiratory mutants impedes the distinction between core and accessory functions. Newly developed synthetic bypasses of photorespiration, beyond holding potential for significant yield increases in C3 crops, will enable us to differentiate between essential and accessory functions of photorespiration.

Cold Acclimation of the Thermoacidophilic Red Alga Galdieria sulphuraria: Changes in Gene Expression and Involvement of Horizontally Acquired Genes.

Galdieria sulphuraria is a unicellular red alga that lives in hot, acidic, toxic metal-rich, volcanic environments, where few other organisms survive. Its genome harbors up to 5% of genes that were most likely acquired through horizontal gene transfer. These genes probably contributed to G.sulphuraria's adaptation to its extreme habitats, resulting in today's polyextremophilic traits. Here, we applied RNA-sequencing to obtain insights into the acclimation of a thermophilic organism towards temperatures below its growth optimum and to study how horizontally acquired genes contribute to cold acclimation. A decrease in growth temperature from 42�C/46�C to 28�C resulted in an upregulation of ribosome biosynthesis, while excreted proteins, probably components of the cell wall, were downregulated. Photosynthesis was suppressed at cold temperatures, and transcript abundances indicated that C-metabolism switched from gluconeogenesis to glycogen degradation. Folate cycle and S-adenosylmethionine cycle (one-carbon metabolism) were transcriptionally upregulated, probably to drive the biosynthesis of betaine. All these cold-induced changes in gene expression were reversible upon return to optimal growth temperature. Numerous genes acquired by horizontal gene transfer displayed temperature-dependent expression changes, indicating that these genes contributed to adaptive evolution in G.sulphuraria.

A unique ferredoxin acts as a player in the low-iron response of photosynthetic organisms.

Iron chronically limits aquatic photosynthesis, especially in marine environments, and the correct perception and maintenance of iron homeostasis in photosynthetic bacteria, including cyanobacteria, is therefore of global significance. Multiple adaptive mechanisms, responsive promoters, and posttranscriptional regulators have been identified, which allow cyanobacteria to respond to changing iron concentrations. However, many factors remain unclear, in particular, how iron status is perceived within the cell. Here we describe a cyanobacterial ferredoxin (Fed2), with a unique C-terminal extension, that acts as a player in iron perception. Fed2 homologs are highly conserved in photosynthetic organisms from cyanobacteria to higher plants, and, although they belong to the plant type ferredoxin family of [2Fe-2S] photosynthetic electron carriers, they are not involved in photosynthetic electron transport. As deletion of fed2 appears lethal, we developed a C-terminal truncation system to attenuate protein function. Disturbed Fed2 function resulted in decreased chlorophyll accumulation, and this was exaggerated in iron-depleted medium, where different truncations led to either exaggerated or weaker responses to low iron. Despite this, iron concentrations remained the same, or were elevated in all truncation mutants. Further analysis established that, when Fed2 function was perturbed, the classical iron limitation marker IsiA failed to accumulate at transcript and protein levels. By contrast, abundance of IsiB, which shares an operon with isiA, was unaffected by loss of Fed2 function, pinpointing the site of Fed2 action in iron perception to the level of posttranscriptional regulation.

The Plastid Envelope CHLOROPLAST MANGANESE TRANSPORTER1 Is Essential for Manganese Homeostasis in Arabidopsis.

The transition metal manganese (Mn) is indispensable for photoautotrophic growth since photosystem II (PSII) employs an inorganic Mn4CaO5 cluster for water splitting. Here, we show that the Arabidopsis membrane protein CHLOROPLAST MANGANESE TRANSPORTER1 (CMT1) is involved in chloroplast Mn homeostasis. CMT1 is the closest homolog of the previously characterized thylakoid Mn transporter PHOTOSYNTHESIS-AFFECTED MUTANT71 (PAM71). In contrast to PAM71, CMT1 resides at the chloroplast envelope and is ubiquitously expressed. Nonetheless, like PAM71, the expression of CMT1 can also alleviate the Mn-sensitive phenotype of yeast mutant Δpmr1. The cmt1 mutant is severely suppressed in growth, chloroplast ultrastructure, and PSII activity owing to a decrease in the amounts of pigments and thylakoid membrane proteins. The importance of CMT1 for chloroplast Mn homeostasis is demonstrated by the significant reduction in chloroplast Mn concentrations in cmt1-1, which exhibited reduced Mn binding in PSII complexes. Moreover, CMT1 expression is downregulated in Mn-surplus conditions. The pam71 cmt1-1double mutant resembles the cmt1-1 single mutant rather than pam71 in most respects. Taken together, our results suggest that CMT1 mediates Mn2+ uptake into the chloroplast stroma, and that CMT1 and PAM71 function sequentially in Mn delivery to PSII across the chloroplast envelope and the thylakoid membrane.

Homology-Directed Repair of a Defective Glabrous Gene in Arabidopsis With Cas9-Based Gene Targeting.

The CRISPR/Cas9 system has emerged as a powerful tool for targeted genome editing in plants and beyond. Double-strand breaks induced by the Cas9 enzyme are repaired by the cell's own repair machinery either by the non-homologous end joining pathway or by homologous recombination (HR). While the first repair mechanism results in random mutations at the double-strand break site, HR uses the genetic information from a highly homologous repair template as blueprint for repair of the break. By offering an artificial repair template, this pathway can be exploited to introduce specific changes at a site of choice in the genome. However, frequencies of double-strand break repair by HR are very low. In this study, we compared two methods that have been reported to enhance frequencies of HR in plants. The first method boosts the repair template availability through the formation of viral replicons, the second method makes use of an in planta gene targeting (IPGT) approach. Additionally, we comparatively applied a nickase instead of a nuclease for target strand priming. To allow easy, visual detection of HR events, we aimed at restoring trichome formation in a glabrous Arabidopsis mutant by repairing a defective glabrous1 gene. Using this efficient visual marker, we were able to regenerate plants repaired by HR at frequencies of 0.12% using the IPGT approach, while both approaches using viral replicons did not yield any trichome-bearing plants.

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.