Mn‐catalase (Alr0998) protects the photosynthetic,nitrogen‐fixing cyanobacterium Anabaena PCC7120 from oxidative stress

Role of the non‐haem, manganese catalase (Mn‐catalase) in oxidative stress tolerance is unknown in cyanobacteria. The ORF alr0998 from the Anabaena PCC7120, which encodes a putative Mn‐catalase, was constitutively overexpressed in Anabaena PCC7120 to generate a recombinant strain, AnKat⁺. The Alr0998 protein could be immunodetected in AnKat⁺ cells and zymographic analysis showed a distinct thermostable catalase activity in the cytosol of AnKat⁺ cells but not in the wild‐type Anabaena PCC7120. The observed catalase activity was insensitive to inhibition by azide indicating that Alr0998 protein is indeed a Mn‐catalase. In response to oxidative stress, the AnKat⁺ showed reduced levels of intracellular ROS which was also corroborated by decreased production of an oxidative stress‐inducible 2‐Cys‐Prx protein. Treatment of wild‐type Anabaena PCC7120 with H₂O₂ caused (i) RNA degradation in vivo, (ii) severe reduction of photosynthetic pigments and CO₂ fixation, (iii) fragmentation and lysis of filaments and (iv) loss of viability. In contrast, the AnKat⁺ strain was protected from all the aforesaid deleterious effect under oxidative stress. This is the first report on protection of an organism from oxidative stress by overexpression of a Mn‐catalase.     

Light-to-dark transitions trigger a transient increase in intracellular Ca2+ modulated by the redox state of the photosynthetic electron transport chain in the cyanobacterium Anabaena sp. PCC7120

Light‐to‐dark transitions represent one of the most crucial environmental stresses that photosynthetic organisms must cope with, since substantial metabolism adaptations are required in order to utilize alternative energy and carbon sources. Although signal transduction systems for changing light regimes are not sufficiently understood, calcium has been implicated in plants as a second messenger in light‐on and light‐off events. Much less is known about light signalling in cyanobacteria, but it has been shown that calcium probably performs similar signalling roles in these organisms and other prokaryotes. Herein it is reported that light‐to‐dark transitions trigger a calcium transient in aequorin expressing Anabaena sp. PCC7120. The magnitude of this transient depends on the fluence rate previously irradiated and can reach a peak height over 2 µm free calcium when the fluence rate of light is around 400 µmol photons s^?1 m^?2. The use of increasing calcium concentration, ethylene glycol‐bis (β‐aminoethylether) N,N,N′,N′‐tetraacetic acid (EGTA), verapamil and trifluoperazine indicated that these transients are originated by a calcium influx probably through verapamil‐sensitive Ca²⁺ channels and are probably modulated by calcium‐binding proteins. Experiments with different light spectral qualities and the photosynthetic inhibitors 3‐(3,4 dichlorophenyl)1,1,dimelthylurea (DCMU) and 3,5‐dibromo‐3‐methyl‐b‐isopropyl‐p‐benzoquinone (DBMIB) indicate that the calcium transient triggered by the light‐to‐dark transition is not coupled to a specific photoreceptor but rather to changes in the redox state of photosynthetic electron transport chain components other than the plastoquinone pool.     

The role of extracellular polysaccharides produced by the terrestrial cyanobacterium Nostoc sp. strain HK-01 in NaCl tolerance

The terrestrial cyanobacterium Nostoc sp. HK-01 was more tolerant to NaCl stress than the aquatic cyanobacterium Anabaena sp. PCC 7120 (also called Nostoc sp. PCC 7120) which is similar to Nostoc sp. HK-01 in phylogeny. We determined the amount of extracellular polysaccharides (capsular and released polysaccharides) from the cells of both strains cultured with or without 200 mM NaCl. The amount of capsular polysaccharides from Nostoc HK-01 reached approximately 65% of the dry weight whereas that from Anabaena PCC 7120 only occupied approximately 18% of the dry weight under NaCl stress. Anabaena PCC 7120 grew well under NaCl stress when both polysaccharides from Nostoc HK-01 were added to the culture. However, Anabaena PCC 7120 barely grew under NaCl stress when both of its polysaccharides were added. Extracellular polysaccharides from Nostoc HK-01 contained abundant fucose and glucuronic acid in comparison with those from Anabaena PCC 7120. Under NaCl stress, the composition ratios of sugars in the extracellular polysaccharides from Anabaena PCC 7120 hardly changed in comparison with those in ordinary culture conditions. By contrast, the composition ratios of sugars in the extracellular polysaccharides from Nostoc HK-01 changed under NaCl stress. These results suggest that the effect of extracellular polysaccharides from Nostoc HK-01 on NaCl tolerance comes from the increased amount of capsular polysaccharides, the sugar composition, and the change of the sugar composition ratio under NaCl stress.     

Phosphatidylinositol‐hydrolyzing phospholipase C4 modulates rice response to salt and drought

Phosphatidylinositol‐specific phospholipase C (PI‐PLC) is involved in stress signalling but its signalling function remains largely unknown in crop plants. Here, we report that the PI‐PLC4 from rice (Oryza sativa cv), OsPLC4, plays a positive role in osmotic stress response. Two independent knockout mutants, plc4‐1 and plc4‐2, exhibited decreased seedling growth and survival rate whereas overexpression of OsPLC4 improved survival rate under high salinity and water deficiency, compared with wild type (WT). OsPLC4 hydrolyses PI, phosphatidylinositol 4‐phosphate (PI4P), and phosphatidylinositol‐4,5‐bisphosphate (PIP₂) to generate diacylglycerol (DAG) in vitro. Knockout of OsPLC4 attenuated salt‐induced increase of phosphatidic acid (PA) whereas overexpression of OsPLC4 decreased the level of PI4P and PIP₂ under salt treatment. Applications of DAG or PA restored the growth defect of plc4‐1 to WT but DAG kinase inhibitor 1 blocked the complementary effect of DAG in plc4‐1 under salt stress. In addition, the loss of OsPLC4 compromised the increase of inositol triphosphate and free cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) and inhibited the induction of genes involved in Ca²⁺ sensor and osmotic stress response to salt stress. The results indicate that OsPLC4 modulates the activity of two signalling pathways, PA and Ca²⁺, to affect rice seedling response to osmotic stress.     

Gene Expression in the Cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120 under Desiccation

The N₂-fixing cyanobacterium Anabaena sp. PCC7120 showed an inherent capacity for desiccation tolerance. A DNA microarray covering almost the entire genome of Anabaena was used to determine the genome-wide gene expression under desiccation. RNA was extracted from cells at intervals starting from early to late desiccation. The pattern of gene expression in DNA fragments was categorized into seven types, which include four types of up-regulated and three types of down-regulated fragments. Validation of the data was carried out by RT-PCR on selected up-regulated DNA fragments and was consistent with the changes in mRNA levels. Our conclusions regarding desiccation tolerance for Anabaena sp. PCC7120 are as follows: (i) Genes for osmoprotectant metabolisms and the K⁺ transporting system are up-regulated from early to mid-desiccation; (ii) genes induced by osmotic, salt, and low-temperature stress are up-regulated under desiccation; (iii) genes for heat shock proteins are up-regulated after mid-desiccation; (iv) genes for photosynthesis and the nitrogen-transporting system are down-regulated during early desiccation; and (v) genes for RNA polymerase and ribosomal protein are down-regulated between the early and the middle phase of desiccation. Profiles of gene expression are discussed in relation to desiccation acclimation.     

Regulation of ultraviolet radiation induced cutaneous photoimmunosuppression by toll-like receptor-4

The open reading frame alr3199 of the nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 was cloned and overexpressed in Escherichia coli. Purified recombinant Alr3199 protein was found to be a functionally active deoxyribonuclease with novel features, such as (1) no homology to typical DNases (2) a Ca²⁺-dependent Nickase activity (3) presence of a di-hemerythrin domain, and (4) requirement of Fe²⁺ conjugated to hemerythrin domains for optimal activity. Both the DNase and Nickase activities were found to be associated with the N-terminal non-hemerythrin region, but were modulated by Fe²⁺ conjugated to the C-terminal hemerythrin region. This is the first report of a hemerythrin protein with DNase activity, tentatively designated as ‘HE-DNase’, and with a possible role in stress-induced DNA damage/repair in Anabaena.     

The effect of short-term low-temperature treatments on gene expression in Arabidopsis correlates with changes in intracellular Ca2+ levels

The role of changes in intracellular calcium ion concentration ([Ca²⁺]_i) in low‐temperature signal transduction in plants has lately been supported by several studies. An analysis to determine whether the low‐temperature‐induced increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) could be correlated with a downstream response such as gene expression was carried out. The induction of the low‐temperature‐regulated gene LTI78 was used as an end point marker of the signal transduction pathway. It was found that this gene is induced by very brief low‐temperature exposures and that the induction does not depend on a continuous exposure to low temperature. By altering the cooling rate, different patterns of the Ca²⁺ response were obtained which could be correlated with different patterns of LTI78 induction. Furthermore, reducing the Ca²⁺ transients by pre‐treatment with the Ca²⁺ channel blocker La³⁺ also led to a reduced level of gene induction. The results show that brief exposures to low temperature results in the onset of a signalling pathway that leads to the induction of gene expression. This indicates the involvement of changes in [Ca²⁺]_cyt in low‐temperature signalling leading to LTI78 expression but the presence of multiple signalling pathways is suggested.     

Iron deficiency influences NtcA‐dependent regulation of fatty acid desaturation and heterocyte envelop formation in Anabaena sp. PCC 7120

In Anabaena sp. PCC 7120, iron is an essential trace element and its availability determines proper functioning of several kinds of metabolisms. Iron deficiency leads to several unavoidable consequences including membrane damage. In the present study, we dealt with the impact of iron deficiency on NtcA (global nitrogen regulator)‐dependent regulation of two important processes, i.e. fatty acid desaturation and heterocyte envelop formation in cyanobacterium Anabaena sp. PCC 7120. In Anabaena sp. PCC 7120, NtcA regulates fatty acid desaturation by regulating enzyme fatty acid desaturases. The NtcA‐based regulation of fatty acid desaturation may be direct or indirect. Furthermore, the expression of genes involved in the heterocyte envelope polysaccharide (HEP) layer formation (hepABCK) and heterocyte‐specific glycolipids (HGLs) synthesis (devH, hglE_A, prpJ and devB) were also under the control of NtcA and reduced under iron deficiency background. The enhanced expression of furA and early downregulation of ntcA under iron deficiency is responsible for reduction in fatty acid desaturation as well as decrease in the expression of genes involved in HEP layer formation and HGL synthesis. Overall results confirmed that iron deficiency influences the NtcA‐based regulation of fatty acid desaturation and heterocyte envelop formation in Anabaena sp. PCC 7120.     

Proteome Analysis of Enriched Heterocysts from Two Hydrogenase Mutants from Anabaena sp. PCC 7120

Cyanobacteria are oxygenic photosynthetic prokaryotes and play a crucial role in the Earth's carbon and nitrogen cycles. The photoautotrophic cyanobacterium Anabaena sp. PCC 7120 has the ability to fix atmospheric nitrogen in heterocysts and produce hydrogen as a byproduct through a nitrogenase. In order to improve hydrogen production, mutants from Anabaena sp. PCC 7120 are constructed by inactivation of the uptake hydrogenase (ΔhupL) and the bidirectional hydrogenase (ΔhoxH) in previous studies. Here the proteomic differences of enriched heterocysts between these mutants cultured in N₂‐fixing conditions are investigated. Using a label‐free quantitative proteomics approach, a total of 2728 proteins are identified and it is found that 79 proteins are differentially expressed in the ΔhupL and 117 proteins in the ΔhoxH variant. The results provide for the first time comprehensive information on proteome regulation of the uptake hydrogenase and the bidirectional hydrogenase, as well as systematic data on the hydrogen related metabolism in Anabaena sp. PCC 7120.     

Calcium-dependent protease of the cyanobacterium Anabaena: molecular cloning and expression of the gene in Escherichia coli, sequencing and site-directed mutagenesis

Summary It has been suggested that a calcium-dependent intracellular protease of the cyanobacterium, Anabaena sp., participates in the differentiation of heterocysts, cells that are specialized for fixation of N₂. Clones of the structural gene (designated prcA) for this protease from Anabaena variabilis strain ATCC 29413 and Anabaena sp. strain PCC 7120 were identified via their expression in Escherichia coli. The prcA gene from A. variabilis was sequenced. The genes of both strains, mutated by insertion of a drug resistance cassette, were returned to these same strains of Anabaena on suicide plasmids. The method of sacB-mediated positive selection for double recombinants was used to achieve replacement of the wild-type prcA genes by the mutated forms. The resulting mutants, which lacked Ca²⁺-dependent protease activity, were not impaired in heterocyst formation and grew on N₂ as sole nitrogen source.