The role of ferritin in enhancing the stress tolerance of grapevine

In order to improve the stress tolerance of grapevine, transgenic plants were produced and regenerated from the anthers of a grapevine rootstock cultivar expressing the ferritin of Medicago sativa. Leaf disks of the plants were exposed to oxidative stress by intracellular ROS production using paraquat, a herbicide known to mediate superoxide radical formation in thylakoid membranes. The Medicago ferritin had a high expression level in the transgenic plants under normal condition, and it further increased about twofold under the applied stress condition. In response to the treatment, the transgenic plants showed enhanced stress tolerance as compared to the untransformed cultivar

The Ability of Cyanobacterial Cells to Restore UV-B Radiation Induced Damage to Photosystem II is Influenced by Photolyase Dependent DNA Repair

Damage of DNA and Photosystem-II are among the most significant effects of UV-B irradiation in photosynthetic organisms. Both damaged DNA and Photosystem-II can be repaired, which represent important defense mechanisms against detrimental UV-B effects. Correlation of Photosystem-II damage and repair with the concurrent DNA damage and repair was investigated in the cyanobacterium Synechocystis PCC6803 using its wild type and a photolyase deficient mutant, which is unable to repair UV-B induced DNA damages. A significant amount of damaged DNA accumulated during UV-B exposure in the photolyase mutant concomitant with decreased Photosystem-II activity and D1 protein amount. The transcript level of psbA3, which is a UV-responsive copy of the psbA gene family encoding the D1 subunit of the Photosystem-II reaction center, is also decreased in the photolyase mutant. The wild-type cells, however, did not accumulate damaged DNA during UV-B exposure, suffered smaller losses of Photosystem-II activity and D1 protein, and maintained higher level of psbA3 transcripts than the photolyase mutant. It is concluded that the repair capacity of Photosystem-II depends on the ability of cells to repair UV-B-damaged DNA through maintaining the transcription of genes, which are essential for protein synthesis-dependent repair of the Photosystem-II reaction center

Biomedical potential of cyanobacteria and algae

Cyanobacteria have appeared on the primordial Earth over three billion years ago and still thrive in most habitats. These photosynthetic microbes have remarkable genetic plasticity and variability and have evolved an amazing arsenal of biochemical pathways that exert defence mechanisms and produce metabolites unique to them. By forming plastids, endosymbiont cyanobacteria contributed to the development of plants. Algae, the simplest plants, thrive in similar habitats and face the same challenges of the ever changing environment as cyanobacteria; and they have maintained similarity to them, with respect to production of unique metabolites and utilizing unique pathways. The exploration of these natural compounds and the biochemical pathways leading to their production provide excellent tools in fighting some major challenge that mankind needs to face in our days. In this contribution we briefly list the benefits that the genetics of these microbes and the produced compounds can offer, with emphasis on possible medical relevance. We mention applications in basic science, industry and agriculture, and list the potentials in medical drug development, therapy and nutrition of some enzymes, polysaccharides, polyphenols, pigments, peptides and lipids, among others, in the current state of the world-wide research on the topic

A unique regulation of the expression of the psbA, psbD, and psbE genes, encoding the D1, D2 and cytochrome b559 subunits of the Photosystem II complex in the chlorophyll d containing cyanobacterium Acaryochloris marina

AbstractPhotosynthetic electron transport, chromatic photoacclimation and expression of the genes encoding the D1, D2, and cytochrome b559 subunits of the Photosystem II complex were studied in the chlorophyll d containing cyanobacterium Acaryochloris marina MBIC11017 under various environmental conditions. During oxygen deprivation and inhibition of photosynthetic electron transport by dibromothymoquinone the psbA1 gene encoding a D1′ isoform was induced. All of the three psbA and one of the three psbD (psbD2) genes, encoding two different isoforms of the D1 and the abundant isoform of the D2 proteins, respectively were induced under exposure to UV-B radiation and high intensity visible light. Under far red light the amount of Photosystem II complexes increased, and expression of the psbE2 gene encoding the alpha-subunit of cytochrome b559 was enhanced. However, the psbF and psbE1 genes encoding the beta- and another isoform of alpha-cytochrome b559, respectively remained lowly expressed under all conditions. Far red light also induced the psbD3 gene encoding a D2′ isoform whose primary structure is different from the abundant D2 isoform. psbD3 was also induced under low intensity visible light, when chromatic photoacclimation was indicated by a red-shifted absorption of chlorophyll d. Our results show that differential expression of multigene families encoding different isoforms of D1 and D2 plays an important role in the acclimation of A. marina to contrasting environmental conditions. Moreover, the disproportionate quantity of transcripts of the alpha and beta subunits of cytochrome b559 implies the existence of an alpha–alpha homodimer organization of cytochrome b559 in Photosystem II complexes

A ferritin szerepe a szőlő stressztűrő képességének fokozásában = The role of ferritin in enhancing the stress tolerance of grapevine

Embriogén kalluszt állítottuk elő Richter110 (alany) és Chardonnay (nemes) szőlő fajták portokajiból. Ezeket Medicago sativa ferritint (MsFerr) tartalmazó Agrobacterium vektorokkal transzformáltunk. A DNS beépülését genomikus PCR-rel ellenőriztük. Poliklonális ellenanyagot állítottunk elő, mellyel megállapítottuk, hogy a transzláció magas szintű volt, a transzkriptumból nagy mennyiségű, megfelelően processzált fehérjetermék keletkezett. A transzgenikus szőlő növények elkészültéig dohányban teszteltük az MsFerr gént tartalmazó konstrukciókat. A teljes növényeken végzett UV-B kezelés, ill. levélkorongok közvetlen oxidatív stresszre adott válaszai alapján megállapítottuk, hogy az MsFerr növények toleránsabbak voltak, mint a nem expresszáló kontrollok. Az MsFerr Richter 110 növények regenerálása, szelekciója és felszaporítása sikeres volt, az MsFerr Chardonnay növények felszaporítása azonban hajtásnövekedési problémák miatt nem sikerült. Három MsFerr Richter 110 vonalat vizsgáltunk. Gyökér stresszként fás szárú dugványok tápoldatához hidrogénkarbonátot adtunk, ami klorotikus és levél száradási tünetek okozott Ebben nem kaptunk lényeges különbséget a transzgenikus és transzformálatlan növények között. Ezzel szemben, a leveleket érő hatásokkal: paraquat, NaCl só-stressz és tBHP indukált lipid peroxidációval szemben az MsFerr expresszáló vonalak toleránsabbak voltak (kisebb stressz-indukált fotoszintézis csökkenést mutattak) mint a transzformálatlanok. | Embryogenic calli were started from anthers of Richter 110 (rootstock) and Chardonnay (scion) grapevine and transformed with Agrobacterium harbouring Medicago sativa ferritin gene (MsFerr). Genomic PCR and protein immunoblotting using a polyclonal antibody confirmed that the transcription and processing of MsFerr was successful. Regeneration, selection and propagation of MsFerr Richter 110 plants was successful, but transformed MsFerr Chardonnay plants did not grow sufficiently. Until transgenic grapevine plants became available, preliminary experiments were carried out with MsFerr expressing tobacco. UV-B irradiation of whole plants as well as treatments of leaf disks with various chemical elicitors showed that MsFerr plants were more stress tolerant than non-expressing controls. After regeneration and propagation, three transgenic MsFerr Richter 110 lines were tested. Roots of green cuttings were stressed by flooding and in this experiment transgenic plants did not show significantly higher tolerance to hypoxia/bicarbonate than non-transferred ones. Leaves, however, showed increased tolerance to paraquat, salt stress and tBHP induced lipid peroxidation: their photosynthesis was less affected by these stressors than those from non-transformed plants

Increased sensitivity of heavy metal bioreporters in transporter deficient Synechocystis PCC6803 mutants

The detection and identification of heavy metal contaminants are becoming increasingly important as environmental pollution causes an ever-increasing health hazard in the last decades. Bacterial heavy metal reporters, which constitute an environmentally friendly and cheap approach, offer great help in this process. Although their application has great potential in the detection of heavy metal contamination, their sensitivity still needs to be improved. In this study, we describe a simple molecular biology approach to improve the sensitivity of bacterial heavy metal biosensors. The constructs are luxAB marker genes regulated by the promoters of heavy metal exporter genes. We constructed a mutant strain lacking the cluster of genes responsible for heavy metal transport and hence achieved increased intracellular heavy metal content of the Synechocystis PCC6803 cyanobacterium. Taking advantage of this increased intracellular heavy metal concentration the Ni(2+); Co(2+) and Zn(2+) detection limits of the constructs were three to tenfold decreased compared to the sensitivity of the same constructs in the wild-type cyanobacterium

Increased sensitivity of heavy metal bioreporters in transporter deficient Synechocystis PCC6803 mutants

The detection and identification of heavy metal contaminants are becoming increasingly important as environmental pollution causes an ever-increasing health hazard in the last decades. Bacterial heavy metal reporters, which constitute an environmentally friendly and cheap approach, offer great help in this process. Although their application has great potential in the detection of heavy metal contamination, their sensitivity still needs to be improved. In this study, we describe a simple molecular biology approach to improve the sensitivity of bacterial heavy metal biosensors. The constructs are luxAB marker genes regulated by the promoters of heavy metal exporter genes. We constructed a mutant strain lacking the cluster of genes responsible for heavy metal transport and hence achieved increased intracellular heavy metal content of the Synechocystis PCC6803 cyanobacterium. Taking advantage of this increased intracellular heavy metal concentration the Ni2+; Co2+ and Zn2+ detection limits of the constructs were three to tenfold decreased compared to the sensitivity of the same constructs in the wild-type cyanobacterium

A simple method to produceSynechocystisPCC6803 biofilm under laboratory conditions for electron microscopic and functional studies

Cyanobacteria can form biofilms in nature, which have ecological roles and high potential for practical applications. In order to study them we need biofilm models that contain healthy cells and can withstand physical manipulations needed for structural studies. At present, combined studies on the structural and physiological features of axenic cyanobacterial biofilms are limited, mostly due to the shortage of suitable model systems. Here, we present a simple method to establish biofilms using the cyanobacteriumSynechocystisPCC6803 under standard laboratory conditions to be directly used for photosynthetic activity measurements and scanning electron microscopy (SEM). We found that glass microfiber filters (GMF) with somewhat coarse surface features provided a suitable skeleton to formSynechocystisPCC6803 biofilms. Being very fragile, untreated GMFs were unable to withstand the processing steps needed for SEM. Therefore, we used polyhydroxybutyrate coating to stabilize the filters. We found that up to five coats resulted in GMF stabilization and made possible to obtain high resolution SEM images of the structure of the surface-attached cells and the extensive exopolysaccharide and pili network, which are essential features of biofilm formation. By using pulse-amplitude modulated variable chlorophyll fluorescence imaging, it was also demonstrated that the biofilms contain photosynthetically active cells. Therefore, theSynechocystisPCC6803 biofilms formed on coated GMFs can be used for both structural and functional investigations. The model presented here is easy to replicate and has a potential for high-throughput studies

A single plasmid based CRISPR interference in Synechocystis 6803 - A proof of concept

We developed a simple method to apply CRISPR interference by modifying an existing plasmid pCRISPathBrick containing the native S. pyogenes CRISPR assembly for Synechocystis PCC6803 and named it pCRPB1010. The technique presented here using deadCas9 is easier to implement for gene silencing in Synechocystis PCC6803 than other existing techniques as it circumvents the genome integration and segregation steps thereby significantly shortens the construction of the mutant strains. We executed CRISPR interference against well characterized photosynthetic genes to get a clear phenotype to validate the potential of pCRPB1010 and presented the work as a "proof of concept". Targeting the non-template strand of psbO gene resulted in decreased amount of PsbO and 50% decrease in oxygen evolution rate. Targeting the template strand of psbA2 and psbA3 genes encoding the D1 subunit of photosystem II (PSII) using a single spacer against the common sequence span of the two genes, resulted in full inhibition of both genes, complete abolition of D1 protein synthesis, complete loss of oxygen evolution as well as photoautotrophic growth arrest. This is the first report of a single plasmid based, completely lesion free and episomal expression and execution of CRISPR interference in Synechocystis PCC6803