Transcriptomic and Proteomic Profiling of Anabaena sp. Strain 90 under Inorganic Phosphorus Stress.

Inorganic phosphorus (Pi) is one of the main growth-limiting factors of diazotrophic cyanobacteria. Due to human activity, the availability of Pi has increased in water bodies, resulting in eutrophication and the formation of massive cyanobacterial blooms. In this study, we examined the molecular responses of the cyanobacterium Anabaena sp. strain 90 to phosphorus deprivation, aiming at the identification of candidate genes to monitor the Pi status in cyanobacteria. Furthermore, this study increased the basic understanding of how phosphorus affects diazotrophic and bloom-forming cyanobacteria as a major growth-limiting factor. Based on RNA sequencing data, we identified 246 differentially expressed genes after phosphorus starvation and 823 differentially expressed genes after prolonged Pi limitation, most of them related to central metabolism and cellular growth. The transcripts of the genes related to phosphorus transport and assimilation (pho regulon) were most upregulated during phosphorus depletion. One of the most increased transcripts encodes a giant protein of 1,869 amino acid residues, which contains, among others, a phytase-like domain. Our findings predict its crucial role in phosphorus starvation, but future studies are still needed. Using two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found 43 proteins that were differentially expressed after prolonged phosphorus stress. However, correlation analysis unraveled an association only to some extent between the transcriptomic and proteomic abundances. Based on the present results, we suggest that the method used for monitoring the Pi status in cyanobacterial bloom should contain wider combinations of pho regulon genes (e.g., PstABCS transport systems) in addition to the commonly used alkaline phosphatase gene alone.

Flavodiiron protein Flv2/Flv4-related photoprotective mechanism dissipates excitation pressure of PSII in cooperation with phycobilisomes in Cyanobacteria.

Oxygenic photosynthesis evolved with cyanobacteria, the ancestors of plant chloroplasts. The highly oxidizing chemistry of water splitting required concomitant evolution of efficient photoprotection mechanisms to safeguard the photosynthetic machinery. The role of flavodiiron proteins (FDPs), originally called A-type flavoproteins or Flvs, in this context has only recently been appreciated. Cyanobacterial FDPs constitute a specific protein group that evolved to protect oxygenic photosynthesis. There are four FDPs in Synechocystis sp. PCC 6803 (Flv1 to Flv4). Two of them, Flv2 and Flv4, are encoded by an operon together with a Sll0218 protein. Their expression, tightly regulated by CO2 levels, is also influenced by changes in light intensity. Here we describe the overexpression of the flv4-2 operon in Synechocystis sp. PCC 6803 and demonstrate that it results in improved photochemistry of PSII. The flv4-2/OE mutant is more resistant to photoinhibition of PSII and exhibits a more oxidized state of the plastoquinone pool and reduced production of singlet oxygen compared with control strains. Results of biophysical measurements indicate that the flv4-2 operon functions in an alternative electron transfer pathway from PSII, and thus alleviates PSII excitation pressure by channeling up to 30% of PSII-originated electrons. Furthermore, intact phycobilisomes are required for stable expression of the flv4-2 operon genes and for the Flv2/Flv4 heterodimer-mediated electron transfer mechanism. The latter operates in photoprotection in a complementary way with the orange carotenoid protein-related nonphotochemical quenching. Expression of the flv4-2 operon and exchange of the D1 forms in PSII centers upon light stress, on the contrary, are mutually exclusive photoprotection strategies among cyanobacteria.

The antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 prevents premature expression of the flv4-2 operon upon shift in inorganic carbon supply.

The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (C(i)), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the Q(B) site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by C(i) limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in C(i) conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon.

Proteomic profiles of thylakoid membranes and changes in response to iron deficiency.

The proteomic profile of thylakoid membranes and the changes induced in that proteome by iron deficiency have been studied by using thylakoid preparations from Beta vulgaris plants grown in hydroponics. Two different 2-D electrophoresis approaches have been used to study these proteomes: isoelectrical focusing followed by SDS PAGE (IEF-SDS PAGE) and blue-native polyacrylamide gel electrophoresis followed by SDS PAGE (BN-SDS PAGE). These techniques resolved approximately 110-140 and 40 polypeptides, respectively. Iron deficiency induced significant changes in the thylakoid sugar beet proteome profiles: the relative amounts of electron transfer protein complexes were reduced, whereas those of proteins participating in leaf carbon fixation-linked reactions were increased. A set of polypeptides, which includes several enzymes related to metabolism, was detected in thylakoid preparations from Fe-deficient Beta vulgaris leaves by using BN-SDS PAGE, suggesting that they may be associated with these thylakoids in vivo. The BN-SDS PAGE technique has been proven to be a better method than IEF-SDS PAGE to resolve highly hydrophobic integral membrane proteins from thylakoid preparations, allowing for the identification of complexes and determination of their polypeptidic components.