A cobalt concentration sensitive Btu-like system facilitates cobalamin uptake in Anabaena sp. PCC 7120.

Metal homeostasis is central to all forms of life, as metals are essential micronutrients with toxic effects at elevated levels. Macromolecular machines facilitate metal uptake into the cells and their intracellular level is regulated by multiple means, which can involve RNA elements and proteinaceous components. While the general principles and components for uptake and cellular content regulation of, e.g., cobalt have been identified for proteobacteria, the corresponding mechanism in other Gram-negative bacteria such as cyanobacteria remain to be established. Based on their photosynthetic activity, cyanobacteria are known to exhibit a special metal demand in comparison to other bacteria. Here, the regulation by cobalt and cobalamin as well as their uptake is described for Anabaena sp. PCC 7120, a model filamentous heterocyst-forming cyanobacterium. Anabaena contains at least three cobalamin riboswitches in its genome, for one of which the functionality is confirmed here. Moreover, two outer membrane-localized cobalamin TonB-dependent transporters, namely BtuB1 and BtuB2, were identified. BtuB2 is important for fast uptake of cobalamin under conditions with low external cobalt, whereas BtuB1 appears to function in cobalamin uptake under conditions of sufficient cobalt supply. While the general function is comparable, the specific function of the two genes differs and mutants thereof show distinct phenotypes. The uptake of cobalamin depends further on the TonB and a BtuFCD machinery, as mutants of tonB3 and btuD show reduced cobalamin uptake rates. Thus, our results provide novel information on the uptake of cobalamin and the regulation of the cellular cobalt content in cyanobacteria.

Two TonB-dependent outer membrane transporters involved in heme uptake in Anabaena sp. PCC 7120.

Low availability of micronutrients such as iron has enforced the evolution of uptake systems in all kingdoms of life. In Gram-negative bacteria, outer membrane, periplasmatic and plasma membrane localized proteins facilitate the uptake of iron-loaded chelators, which are energized by TonB proteins. The specificity of different uptake systems likely depends either on the endogenously produced siderophore or on the bioavailability of iron-chelator complexes in the environment. Hence, an uptake system for schizokinen produced by the model cyanobacterium Anabaena sp. PCC 7120 exists, while bioinformatics analysis suggests the existence of additional systems, likely for uptake of xenosiderophores. Consistently, proteins encoded by alr2153 (hutA1) and alr3242 (hutA2) are assigned as outer membrane heme transporters. Indeed, Anabaena sp. PCC 7120 can utilize external heme as an iron source. The addition of heme resulted in an induction of genes involved in heme degradation and chlorophyll a synthesis and in an increase of the chlorophyll a content. Moreover, iron starvation induced the expression of hutA1, while the addition of heme led to its repression. Remarkably, the addition of a high concentration of heme but not iron starvation resulted in hutA2 induction. Plasmid insertion mutants of both genes exhibited a reduced capacity to recover from iron starvation by heme addition, which indicates a dependence of heme uptake on functional HutA1 and HutA2 proteins. The structural model generated by bioinformatics methods is further in agreement with a role in heme uptake. Thus, we provide evidence that Anabaena sp. PCC 7120 uses a heme uptake system in parallel to other iron acquisition systems.

Low dose ribosomal DNA P-loop mutation affects development and enforces autophagy in Arabidopsis.

Arabidopsis contains hundreds of ribosomal DNA copies organized within the nucleolar organizing regions (NORs) in chromosomes 2 and 4. There are four major types of variants of rDNA, VAR1-4, based on the polymorphisms of 3' external transcribed sequences. The variants are known to be differentially expressed during plant development. We created a mutant by the CRISPR-Cas9-mediated excision of ~ 25 nt from predominantly NOR4 ribosomal DNA copies, obtaining mosaic mutational events on ~ 5% of all rDNA copies. The excised region consists of P-loop and Helix-82 segments of 25S rRNA. The mutation led to allelic, dosage-dependent defects marked by lateral root inhibition, reduced size, and pointy leaves, all previously observed for defective ribosomal function. The mutation in NOR4 led to dosage compensation from the NOR2 copies by elevated expression of VAR1 in mutants and further associated single-nucleotide variants, thus, resulting in altered rRNA sub-population. Furthermore, the mutants exhibited rRNA maturation defects specifically in the minor pathway typified by 32S pre-rRNA accumulation. Density-gradient fractionation and subsequent RT-PCR of rRNA analyses revealed that mutated copies were not incorporated into the translating ribosomes. The mutants in addition displayed an elevated autophagic flux as shown by the autophagic marker GFP-ATG8e, likely related to ribophagy.

Cyclophilin anaCyp40 regulates photosystem assembly and phycobilisome association in a cyanobacterium.

Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins.

Enhanced pro-apoptosis gene signature following the activation of TAp63α in oocytes upon γ irradiation.

Specialized surveillance mechanisms are essential to maintain the genetic integrity of germ cells, which are not only the source of all somatic cells but also of the germ cells of the next generation. DNA damage and chromosomal aberrations are, therefore, not only detrimental for the individual but affect the entire species. In oocytes, the surveillance of the structural integrity of the DNA is maintained by the p53 family member TAp63α. The TAp63α protein is highly expressed in a closed and inactive state and gets activated to the open conformation upon the detection of DNA damage, in particular DNA double-strand breaks. To understand the cellular response to DNA damage that leads to the TAp63α triggered oocyte death we have investigated the RNA transcriptome of oocytes following irradiation at different time points. The analysis shows enhanced expression of pro-apoptotic and typical p53 target genes such as CDKn1a or Mdm2, concomitant with the activation of TAp63α. While DNA repair genes are not upregulated, inflammation-related genes become transcribed when apoptosis is initiated by activation of STAT transcription factors. Furthermore, comparison with the transcriptional profile of the ΔNp63α isoform from other studies shows only a minimal overlap, suggesting distinct regulatory programs of different p63 isoforms.

Structural analysis of temperature-dependent alternative splicing of HsfA2 pre-mRNA from tomato plants.

Temperature-dependent alternative splicing was recently demonstrated for intron 2 of the gene coding for heat shock factor HsfA2 of the tomato plant Solanum lycopersicum, but the molecular mechanism regulating the abundance of such temperature-dependent splice variants is still unknown. We report here on regulatory pre-mRNA structures that could function as regulators by controlling the use of splice sites in a temperature-dependent manner. We investigate pre-mRNA structures at the splice sites of intron 2 of the gene coding for HsfA2 from S. lycopersicum using NMR- and CD-spectroscopy as well as in-line probing. The pre-mRNA undergoes conformational changes between two different secondary structures at the 3' splice site of the intron in a temperature-dependent manner. Previously, it was shown that three single nucleotide polymorphisms (SNPs) in intron 2 of the HsfA2 pre-mRNA affect the splicing efficiency of its pre-mRNA and are linked to the thermotolerance in different tomato species. By comparing pre-mRNA fragments of the tomato species S. lycopersicum and S. peruvianum, we show that these SNPs result in substantial structural differences between the pre-mRNAs of the two species.

Retrograde Analysis of Calcium Signaling by CaMPARI2 Shows Cytosolic Calcium in Chondrocytes Is Unaffected by Parabolic Flights.

Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.

HsfA7 coordinates the transition from mild to strong heat stress response by controlling the activity of the master regulator HsfA1a in tomato.

Plants respond to higher temperatures by the action of heat stress (HS) transcription factors (Hsfs), which control the onset, early response, and long-term acclimation to HS. Members of the HsfA1 subfamily, such as tomato HsfA1a, are the central regulators of HS response, and their activity is fine-tuned by other Hsfs. We identify tomato HsfA7 as capacitor of HsfA1a during the early HS response. Upon a mild temperature increase, HsfA7 is induced in an HsfA1a-dependent manner. The subsequent interaction of the two Hsfs prevents the stabilization of HsfA1a resulting in a negative feedback mechanism. Under prolonged or severe HS, HsfA1a and HsfA7 complexes stimulate the induction of genes required for thermotolerance. Therefore, HsfA7 exhibits a co-repressor mode at mild HS by regulating HsfA1a abundance to moderate the upregulation of HS-responsive genes. HsfA7 undergoes a temperature-dependent transition toward a co-activator of HsfA1a to enhance the acquired thermotolerance capacity of tomato plants.

Functional Diversity of TonB-Like Proteins in the Heterocyst-Forming Cyanobacterium Anabaena sp. PCC 7120.

The TonB-dependent transport of scarcely available substrates across the outer membrane is a conserved feature in Gram-negative bacteria. The plasma membrane-embedded TonB-ExbB-ExbD accomplishes complex functions as an energy transducer by physically interacting with TonB-dependent outer membrane transporters (TBDTs). TonB mediates structural rearrangements in the substrate-loaded TBDTs that are required for substrate translocation into the periplasm. In the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, four TonB-like proteins have been identified. Out of these TonB3 accomplishes the transport of ferric schizokinen, the siderophore which is secreted by Anabaena to scavenge iron. In contrast, TonB1 (SjdR) is exceptionally short and not involved in schizokinen transport. The proposed function of SjdR in peptidoglycan structuring eliminates the protein from the list of TonB proteins in Anabaena. Compared with the well-characterized properties of SjdR and TonB3, the functions of TonB2 and TonB4 are yet unknown. Here, we examined tonB2 and tonB4 mutants for siderophore transport capacities and other specific phenotypic features. Both mutants were not or only slightly affected in schizokinen transport, whereas they showed decreased nitrogenase activity in apparently normal heterocysts. Moreover, the cellular metal concentrations and pigment contents were altered in the mutants, most pronouncedly in the tonB2 mutant. This strain showed an altered susceptibility toward antibiotics and SDS and formed cell aggregates when grown in liquid culture, a phenotype associated with an elevated lipopolysaccharide (LPS) production. Thus, the TonB-like proteins in Anabaena appear to take over distinct functions, and the mutation of TonB2 strongly influences outer membrane integrity. IMPORTANCE The genomes of many organisms encode more than one TonB protein, and their number does not necessarily correlate with that of TonB-dependent outer membrane transporters. Consequently, specific as well as redundant functions of the different TonB proteins have been identified. In addition to a role in uptake of scarcely available nutrients, including iron complexes, TonB proteins are related to virulence, flagellum assembly, pilus localization, or envelope integrity, including antibiotic resistance. The knowledge about the function of TonB proteins in cyanobacteria is limited. Here, we compare the four TonB proteins of Anabaena sp. strain PCC 7120, providing evidence that their functions are in part distinct, since mutants of these proteins exhibit specific features but also show some common impairments.

Dynamics and thermal sensitivity of ribosomal RNA maturation paths in plants.

Ribosome biogenesis is a constitutive fundamental process for cellular function. Its rate of production depends on the rate of maturation of precursor ribosomal RNA (pre-rRNA). The rRNA maturation paths are marked by four dominant rate-limiting intermediates with cell-type variation of the processivity rate. We have identified that high temperature stress in plants, while halting the existing pre-rRNA maturation schemes, also transiently triggers an atypical pathway for 35S pre-rRNA processing. This pathway leads to production of an aberrant precursor rRNA, reminiscent of yeast 24S, encompassing 18S and 5.8S rRNA that do not normally co-occur together at sub-unit levels; this response is elicited specifically by high and not low temperatures. We show this response to be conserved in two other model crop plant species (Rice and Tomato). This pathway persists even after returning to normal growth conditions for 1 hour and is reset between 1-6 hours after stress treatment, likely, due to resumption of normal 35S pre-rRNA synthesis and processing. The heat-induced ITS2 cleavage-derived precursors and stalled P-A2-like precursors were heterogeneous in nature with a fraction containing polymeric (A) tails. Furthermore, high temperature treatment and subsequent fractionation resulted in polysome and precursor rRNA depletion.

The Arabidopsis 2'-O-Ribose-Methylation and Pseudouridylation Landscape of rRNA in Comparison to Human and Yeast.

Eukaryotic ribosome assembly starts in the nucleolus, where the ribosomal DNA (rDNA) is transcribed into the 35S pre-ribosomal RNA (pre-rRNA). More than two-hundred ribosome biogenesis factors (RBFs) and more than two-hundred small nucleolar RNAs (snoRNA) catalyze the processing, folding and modification of the rRNA in Arabidopsis thaliana. The initial pre-ribosomal 90S complex is formed already during transcription by association of ribosomal proteins (RPs) and RBFs. In addition, small nucleolar ribonucleoprotein particles (snoRNPs) composed of snoRNAs and RBFs catalyze the two major rRNA modification types, 2'-O-ribose-methylation and pseudouridylation. Besides these two modifications, rRNAs can also undergo base methylations and acetylation. However, the latter two modifications have not yet been systematically explored in plants. The snoRNAs of these snoRNPs serve as targeting factors to direct modifications to specific rRNA regions by antisense elements. Today, hundreds of different sites of modifications in the rRNA have been described for eukaryotic ribosomes in general. While our understanding of the general process of ribosome biogenesis has advanced rapidly, the diversities appearing during plant ribosome biogenesis is beginning to emerge. Today, more than two-hundred RBFs were identified by bioinformatics or biochemical approaches, including several plant specific factors. Similarly, more than two hundred snoRNA were predicted based on RNA sequencing experiments. Here, we discuss the predicted and verified rRNA modification sites and the corresponding identified snoRNAs on the example of the model plant Arabidopsis thaliana. Our summary uncovers the plant modification sites in comparison to the human and yeast modification sites.

A TonB-Like Protein, SjdR, Is Involved in the Structural Definition of the Intercellular Septa in the Heterocyst-Forming Cyanobacterium Anabaena.

Cyanobacteria are photosynthetic organisms with a Gram-negative envelope structure. Certain filamentous species such as Anabaena sp. strain PCC 7120 can fix dinitrogen upon depletion of combined nitrogen. Because the nitrogen-fixing enzyme, nitrogenase, is oxygen sensitive, photosynthesis and nitrogen fixation are spatially separated in Anabaena. Nitrogen fixation takes place in specialized cells called heterocysts, which differentiate from vegetative cells. During heterocyst differentiation, a microoxic environment is created by dismantling photosystem II and restructuring the cell wall. Moreover, solute exchange between the different cell types is regulated to limit oxygen influx into the heterocyst. The septal zone containing nanopores for solute exchange is constricted between heterocysts and vegetative cells, and cyanophycin plugs are located at the heterocyst poles. We identified a protein previously annotated as TonB1 that is largely conserved among cyanobacteria. A mutant of the encoding gene formed heterocysts but was impaired in diazotrophic growth. Mutant heterocysts appeared elongated and exhibited abnormal morphological features, including a reduced cyanophycin plug, an enhanced septum size, and a restricted nanopore zone in the septum. In spite of this, the intercellular transfer velocity of the fluorescent marker calcein was increased in the mutant compared to the wild type. Thus, the protein is required for proper formation of septal structures, expanding our emerging understanding of Anabaena peptidoglycan plasticity and intercellular solute exchange, and is therefore renamed SjdR (septal junction disk regulator). Notably, calcium supplementation compensated for the impaired diazotrophic growth and alterations in septal peptidoglycan in the sjdR mutant, emphasizing the importance of calcium for cell wall structure. IMPORTANCE Multicellularity in bacteria confers an improved adaptive capacity to environmental conditions and stresses. This includes an enhanced capability of resource utilization through a distribution of biochemical processes between constituent cells. This specialization results in a mutual dependency of different cell types, as is the case for nitrogen-fixing heterocysts and photosynthetically active vegetative cells in Anabaena. In this cyanobacterium, intercellular solute exchange is facilitated through nanopores in the peptidoglycan between adjacent cells. To ensure functionality of the specialized cells, septal size as well as the position, size, and frequency of nanopores in the septum need to be tightly established. The novel septal junction disk regulator SjdR characterized here is conserved in the cyanobacterial phylum. It influences septal size and septal nanopore distribution. Consequently, its absence severely affects the intercellular communication and the strains' growth capacity under nitrogen depletion. Thus, SjdR is involved in septal structure remodeling in cyanobacteria.

Comparative Phenotypic Analysis of Anabaena sp. PCC 7120 Mutants of Porinlike Genes.

Porins are essential for the viability of Gram-negative bacteria. They ensure the uptake of nutrients, can be involved in the maintenance of outer membrane integrity and define the antibiotic or drug resistance of organisms. The function and structure of porins in proteobacteria is well described, while their function in photoautotrophic cyanobacteria has not been systematically explored. We compared the domain architecture of nine putative porins in the filamentous cyanobacterium Anabaena sp. PCC 7120 and analyzed the seven candidates with predicted OprB-domain. Single recombinant mutants of the seven genes were created and their growth capacity under different conditions was analyzed. Most of the putative porins seem to be involved in the transport of salt and copper, as respective mutants were resistant to elevated concentrations of these substances. In turn, only the mutant of alr2231 was less sensitive to elevated zinc concentrations, while mutants of alr0834, alr4741 and all4499 were resistant to high manganese concentrations. Notably the mutant of alr4550 shows a high sensitivity against harmful compounds, which is indicative for a function related to the maintenance of outer membrane integrity. Moreover, the mutant of all5191 exhibited a phenotype which suggests either a higher nitrate demand or an inefficient nitrogen fixation. The dependency of porin membrane insertion on Omp85 proteins was tested exemplarily for Alr4550, and an enhanced aggregation of Alr4550 was observed in two omp85 mutants. The comparative analysis of porin mutants suggests that the proteins in parts perform distinct functions related to envelope integrity and solute uptake.

Identification and Regulation of Tomato Serine/Arginine-Rich Proteins Under High Temperatures.

Alternative splicing is an important mechanism for the regulation of gene expression in eukaryotes during development, cell differentiation or stress response. Alterations in the splicing profiles of genes under high temperatures that cause heat stress (HS) can impact the maintenance of cellular homeostasis and thermotolerance. Consequently, information on factors involved in HS-sensitive alternative splicing is required to formulate the principles of HS response. Serine/arginine-rich (SR) proteins have a central role in alternative splicing. We aimed for the identification and characterization of SR-coding genes in tomato (Solanum lycopersicum), a plant extensively used in HS studies. We identified 17 canonical SR and two SR-like genes. Several SR-coding genes show differential expression and altered splicing profiles in different organs as well as in response to HS. The transcriptional induction of five SR and one SR-like genes is partially dependent on the master regulator of HS response, HS transcription factor HsfA1a. Cis-elements in the promoters of these SR genes were predicted, which can be putatively recognized by HS-induced transcription factors. Further, transiently expressed SRs show reduced or steady-state protein levels in response to HS. Thus, the levels of SRs under HS are regulated by changes in transcription, alternative splicing and protein stability. We propose that the accumulation or reduction of SRs under HS can impact temperature-sensitive alternative splicing.

Insertion of plastidic ß-barrel proteins into the outer envelopes of plastids involves an intermembrane space intermediate formed with Toc75-V/OEP80.

The insertion of organellar membrane proteins with the correct topology requires the following: First, the proteins must contain topogenic signals for translocation across and insertion into the membrane. Second, proteinaceous complexes in the cytoplasm, membrane, and lumen of organelles are required to drive this process. Many complexes required for the intracellular distribution of membrane proteins have been described, but the signals and components required for the insertion of plastidic ß-barrel-type proteins into the outer membrane are largely unknown. The discovery of common principles is difficult, as only a few plastidic ß-barrel proteins exist. Here, we provide evidence that the plastidic outer envelope ß-barrel proteins OEP21, OEP24, and OEP37 from pea (Pisum sativum) and Arabidopsis thaliana contain information defining the topology of the protein. The information required for the translocation of pea proteins across the outer envelope membrane is present within the six N-terminal ß-strands. This process requires the action of translocon of the outer chloroplast (TOC) membrane. After translocation into the intermembrane space, ß-barrel proteins interact with TOC75-V, as exemplified by OEP37 and P39, and are integrated into the membrane. The membrane insertion of plastidic ß-barrel proteins is affected by mutation of the last ß-strand, suggesting that this strand contributes to the insertion signal. These findings shed light on the elements and complexes involved in plastidic ß-barrel protein import.

The Two TpsB-Like Proteins in Anabaena sp. Strain PCC 7120 Are Involved in Secretion of Selected Substrates.

The outer membrane of Gram-negative bacteria acts as an initial diffusion barrier that shields the cell from the environment. It contains many membrane-embedded proteins required for functionality of this system. These proteins serve as solute and lipid transporters or as machines for membrane insertion or secretion of proteins. The genome of Anabaena sp. strain PCC 7120 codes for two outer membrane transporters termed TpsB1 and TpsB2. They belong to the family of the two-partner secretion system proteins which are characteristic of pathogenic bacteria. Because pathogenicity of Anabaena sp. strain PCC 7120 has not been reported, the function of these two cyanobacterial TpsB proteins was analyzed. TpsB1 is encoded by alr1659, while TpsB2 is encoded by all5116 The latter is part of a genomic region containing 11 genes encoding TpsA-like proteins. However, tpsB2 is transcribed independently of a tpsA gene cluster. Bioinformatics analysis revealed the presence of at least 22 genes in Anabaena sp. strain PCC 7120 putatively coding for substrates of the TpsB system, suggesting a rather global function of the two TpsB proteins. Insertion of a plasmid into each of the two genes resulted in altered outer membrane integrity and antibiotic resistance. In addition, the expression of genes coding for the Clp and Deg proteases is dysregulated in these mutants. Moreover, for two of the putative substrates, a dependence of the secretion on functional TpsB proteins could be confirmed. We confirm the existence of a two-partner secretion system in Anabaena sp. strain PCC 7120 and predict a large pool of putative substrates.IMPORTANCE Cyanobacteria are important organisms for the ecosystem, considering their contribution to carbon fixation and oxygen production, while at the same time some species produce compounds that are toxic to their environment. As a consequence, cyanobacterial overpopulation might negatively impact the diversity of natural communities. Thus, a detailed understanding of cyanobacterial interaction with the environment, including other organisms, is required to define their impact on ecosystems. While two-partner secretion systems in pathogenic bacteria are well known, we provide a first description of the cyanobacterial two-partner secretion system.

The Existence and Localization of Nuclear snoRNAs in Arabidopsis thaliana Revisited.

Ribosome biogenesis is one cell function-defining process. It depends on efficient transcription of rDNAs in the nucleolus as well as on the cytosolic synthesis of ribosomal proteins. For newly transcribed rRNA modification and ribosomal protein assembly, so-called small nucleolar RNAs (snoRNAs) and ribosome biogenesis factors (RBFs) are required. For both, an inventory was established for model systems like yeast and humans. For plants, many assignments are based on predictions. Here, RNA deep sequencing after nuclei enrichment was combined with single molecule species detection by northern blot and in vivo fluorescence in situ hybridization (FISH)-based localization studies. In addition, the occurrence and abundance of selected snoRNAs in different tissues were determined. These approaches confirm the presence of most of the database-deposited snoRNAs in cell cultures, but some of them are localized in the cytosol rather than in the nucleus. Further, for the explored snoRNA examples, differences in their abundance in different tissues were observed, suggesting a tissue-specific function of some snoRNAs. Thus, based on prediction and experimental confirmation, many plant snoRNAs can be proposed, while it cannot be excluded that some of the proposed snoRNAs perform alternative functions than are involved in rRNA modification.

miRNAs involved in transcriptome remodeling during pollen development and heat stress response in Solanum lycopersicum.

Cellular transitions during development and stress response depend on coordinated transcriptomic and proteomic alterations. Pollen is particular because its development is a complex process that includes meiotic and mitotic divisions which causes a high heat sensitivity of these cells. Development and stress response are accompanied by a reprogramming of the transcriptome, e.g. by post-transcriptional regulation via miRNAs. We identified known and potentially novel miRNAs in the transcriptome of developing and heat-stressed pollen of Solanum lycopersicum (tomato). The prediction of target mRNAs yielded an equal number of predicted target-sites in CDS and 3'UTR regions of target mRNAs. The result enabled the postulation of a possible link between miRNAs and a fine-tuning of transcription factor abundance during pollen development. miRNAs seem to play a role in the pollen heat stress response as well. We identified several heat stress transcription factors and heat shock proteins as putative targets of miRNAs in response to heat stress, thereby placing these miRNAs as important elements of thermotolerance. Moreover, for members of the AP2, SBP and ARF family members we could predict a miRNA-mediated regulation during development via the miR172, mir156 and mir160-family strengthening the current concept of a cross-connection between development and stress response in plants.

LptC from Anabaena sp. PCC 7120: Expression, purification and crystallization.

Lipopolysaccharides are central elements of the outer leaflet of the outer membrane of Gram-negative bacteria and as such, of cyanobacteria. In the past, the structural analysis of the system in proteobacteria like Escherichia coli has contributed to a deep understanding of the transport of lipopolysaccharides from plasma membrane to the outer membrane. While many components of the transport system are conserved between proteobacteria and cyanobacteria, the periplasmic LptC appears to be distinct. The cyanobacterial proteins are twice as long as the proteobacterial proteins or proteins from firmicutes. This prompted the question whether the structure of the cyanobacterial proteins is comparable the one of the proteobacterial proteins. To address this question, we expressed LptC from Anabaena sp. PCC 7120 in E. coli as truncated protein without the transmembrane segment. We purified the protein utilizing HIS-tag based affinity chromatography and polished the protein after removal of the tag by size exclusion chromatography. The purified recombinant protein was crystallized by the sitting-drop vapor diffusion technique and best crystals, despite being twinned, diffracted to a resolution of 2.6 Å.

Transcriptional Basis for Differential Thermosensitivity of Seedlings of Various Tomato Genotypes.

Transcriptional reprograming after the exposure of plants to elevated temperatures is a hallmark of stress response which is required for the manifestation of thermotolerance. Central transcription factors regulate the stress survival and recovery mechanisms and many of the core responses controlled by these factors are well described. In turn, pathways and specific genes contributing to variations in the thermotolerance capacity even among closely related plant genotypes are not well defined. A seedling-based assay was developed to directly compare the growth and transcriptome response to heat stress in four tomato genotypes with contrasting thermotolerance. The conserved and the genotype-specific alterations of mRNA abundance in response to heat stress were monitored after exposure to three different temperatures. The transcripts of the majority of genes behave similarly in all genotypes, including the majority of heat stress transcription factors and heat shock proteins, but also genes involved in photosynthesis and mitochondrial ATP production. In turn, genes involved in hormone and RNA-based regulation, such as auxin- and ethylene-related genes, or transcription factors like HsfA6b, show a differential regulation that associates with the thermotolerance pattern. Our results provide an inventory of genes likely involved in core and genotype-dependent heat stress response mechanisms with putative role in thermotolerance in tomato seedlings.