Fluorescence quenching of IsiA in early stage of iron deficiency and at cryogenic temperatures

Cyanobacteria respond to iron deficiency during growth by expressing the isiA gene, which produces a chlorophyll-carotenoid protein complex known as IsiA or CP43'. Long-term iron deficiency results in the formation of large IsiA aggregates, some of which associate with photosystem I (PSI) while others are not connected to a photosystem. The fluorescence at room temperature of these unconnected aggregates is strongly quenched, which points to a photoprotective function. In this study, we report time-resolved fluorescence measurements of IsiA aggregates at low temperatures. The average fluorescence lifetimes are estimated to be about 600 ps at 5 K and 150 ps at 80 K. Both lifetimes are much shorter than that of the monomeric complex CP47 at 77 K. We conclude that IsiA aggregates quench fluorescence to a significant extent at cryogenic temperatures. We show by low-temperature fluorescence spectroscopy that unconnected IsiA is present already after two days of growth in an iron-deficient medium, when PSI and PSII are still present in significant amounts and that under these conditions the fluorescence quenching is similar to that after 18 days, when PSI is almost completely absent. We conclude that unconnected IsiA provides photoprotection in all stages of iron deficiency.     

Supramolecular organization and dual function of the IsiA chlorophyll-binding protein in cyanobacteria

A significant part of global primary productivity is provided by cyanobacteria, which are abundant in most marine and freshwater habitats. In many oceanographic regions, however, the concentration of iron can be so low that it limits growth. Cyanobacteria respond to this condition by expressing a number of iron stress inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43'. It was recently shown that 18 IsiA proteins encircle trimeric photosystem I (PSI) under iron-deficient growth conditions. We report here that after prolonged growth of Synechocystis PCC 6803 in an iron-deficient medium, the number of bound IsiA proteins can be much higher than previously known. The largest complexes bind 12-14 units in an inner ring and 19-21 units in an outer ring around a PSI monomer. Fluorescence excitation spectra indicate an efficient light harvesting function for all PSI-bound chlorophylls. We also find that IsiA accumulates in cyanobacteria in excess of what is needed for functional light harvesting by PSI, and that a significant part of IsiA builds supercomplexes without PSI. Because the further decline of PSI makes photosystem II (PSII) increasingly vulnerable to photooxidation, we postulate that the surplus synthesis of IsiA shields PSII from excess light. We suggest that IsiA plays a surprisingly versatile role in cyanobacteria, by significantly enhancing the light harvesting ability of PSI and providing photoprotection for PSII.     

Aggregates of the chlorophyll-binding protein IsiA (CP43') dissipate energy in cyanobacteria

In many natural habitats, growth of cyanobacteria may be limited by a low concentration of iron. Cyanobacteria respond to this condition by expressing a number of iron-stress-inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43'. IsiA monomers assemble into ring-shaped polymers that encircle trimeric or monomeric photosystem I (PSI), or are present in supercomplexes without PSI, in particular upon prolonged iron starvation. In this report, we present steady-state and time-resolved fluorescence measurements of isolated IsiA aggregates that have been purified from an iron-starved psaFJ-minus mutant of Synechocystis PCC 6803. We show that these aggregates have a fluorescence quantum yield of approximately 2% compared to that of chlorophyll a in acetone, and that the dominating fluorescence lifetimes are 66 and 210 ps, more than 1 order of magnitude shorter than that of free chlorophyll a. Comparison of the temperature dependence of the fluorescence yields and spectra of the isolated aggregates and of the cells from which they were obtained suggests that these aggregates occur naturally in the iron-starved cells. We suggest that IsiA aggregates protect cyanobacterial cells against the deleterious effects of light.     

Light-induced energy dissipation in iron-starved cyanobacteria: roles of OCP and IsiA proteins

In response to iron deficiency, cyanobacteria synthesize the iron stress-induced chlorophyll binding protein IsiA. This protein protects cyanobacterial cells against iron stress. It has been proposed that the protective role of IsiA is related to a blue light-induced nonphotochemical fluorescence quenching (NPQ) mechanism. In iron-replete cyanobacterial cell cultures, strong blue light is known to induce a mechanism that dissipates excess absorbed energy in the phycobilisome, the extramembranal antenna of cyanobacteria. In this photoprotective mechanism, the soluble Orange Carotenoid Protein (OCP) plays an essential role. Here, we demonstrate that in iron-starved cells, blue light is unable to quench fluorescence in the absence of the phycobilisomes or the OCP. By contrast, the absence of IsiA does not affect the induction of fluorescence quenching or its recovery. We conclude that in cyanobacteria grown under iron starvation conditions, the blue light-induced nonphotochemical quenching involves the phycobilisome OCP-related energy dissipation mechanism and not IsiA. IsiA, however, does seem to protect the cells from the stress generated by iron starvation, initially by increasing the size of the photosystem I antenna. Subsequently, the IsiA converts the excess energy absorbed by the phycobilisomes into heat through a mechanism different from the dynamic and reversible light-induced NPQ processes.     

THE CYANOBACTERIAL CHLOROPHYLL‐BINDING‐PROTEIN ISIA ACTS TO INCREASE THE IN VIVO EFFECTIVE ABSORPTION CROSS‐SECTION OF PSI UNDER IRON LIMITATION1

Iron availability limits primary production in >30% of the world’s oceans; hence phytoplankton have developed acclimation strategies. In particular, cyanobacteria express IsiA (iron‐stress‐induced) under iron stress, which can become the most abundant chl‐binding protein in the cell. Within iron‐limited oceanic regions with significant cyanobacterial biomass, IsiA may represent a significant fraction of the total chl. We spectroscopically measured the effective cross‐section of the photosynthetic reaction center PSI (σ_PSI) in vivo and biochemically quantified the absolute abundance of PSI, PSII, and IsiA in the model cyanobacterium Synechocystis sp. PCC 6803. We demonstrate that accumulation of IsiA results in a ～60% increase in σ_PSI, in agreement with the theoretical increase in cross‐section based on the structure of the biochemically isolated IsiA‐PSI supercomplex from cyanobacteria. Deriving a chl budget, we suggest that IsiA plays a primary role as a light‐harvesting antenna for PSI. On progressive iron‐stress in culture, IsiA continues to accumulate without a concomitant increase in σ_PSI, suggesting that there may be a secondary role for IsiA. In natural populations, the potential physiological significance of the uncoupled pool of IsiA remains to be established. However, the functional role as a PSI antenna suggests that a large fraction of IsiA‐bound chl is directly involved in photosynthetic electron transport.     

Mobility of the IsiA chlorophyll-binding protein in cyanobacterial thylakoid membranes

We are using fluorescence recovery after photobleaching (FRAP) to probe the dynamics of thylakoid membranes in vivo in cells of the cyanobacterium Synechococcus sp. PCC7942. We have shown previously that the light-harvesting phycobilisomes diffuse quite rapidly on the thylakoid membrane surface. However, the photosystem II core complexes appear completely immobile. This raises the possibility that all of the membrane integral protein complexes in the thylakoid membrane are locked into a rather rigid array. Alternatively, it is possible that photosystem II is specifically anchored in the membrane, with other membrane proteins able to diffuse around it. We have now resolved this question by studying the diffusion of a second integral membrane protein, the IsiA chlorophyll-binding protein. IsiA is induced under iron starvation and some other stress conditions. In iron-stressed cyanobacterial cells, a high proportion of chlorophyll fluorescence comes from IsiA. This makes it straightforward to examine the diffusion of IsiA by FRAP. We find that the complex is mobile with a mean diffusion coefficient of approximately 3 x 10(-11) cm(2) s(-1). Thus it is clear that some thylakoid membrane proteins are mobile and that there must be a specific anchor that prevents photosystem II diffusion. We discuss the implications for the structure and function of the cyanobacterial thylakoid membrane.     

Photophysiological and Photosynthetic Complex Changes during Iron Starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942

Iron is an essential component in many protein complexes involved in photosynthesis, but environmental iron availability is often low as oxidized forms of iron are insoluble in water. To adjust to low environmental iron levels, cyanobacteria undergo numerous changes to balance their iron budget and mitigate the physiological effects of iron depletion. We investigated changes in key protein abundances and photophysiological parameters in the model cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 over a 120 hour time course of iron deprivation. The iron stress induced protein (IsiA) accumulated to high levels within 48 h of the onset of iron deprivation, reaching a molar ratio of ∼42 IsiA : Photosystem I in Synechococcus PCC 7942 and ∼12 IsiA : Photosystem I in Synechocystis PCC 6803. Concomitantly the iron-rich complexes Cytochrome b₆f and Photosystem I declined in abundance, leading to a decrease in the Photosystem I : Photosystem II ratio. Chlorophyll fluorescence analyses showed a drop in electron transport per Photosystem II in Synechococcus, but not in Synechocystis after iron depletion. We found no evidence that the accumulated IsiA contributes to light capture by Photosystem II complexes.     

Fluorescence quenching of IsiA in early stage of iron deficiency and at cryogenic temperatures

Cyanobacteria respond to iron deficiency during growth by expressing the isiA gene, which produces a chlorophyll-carotenoid protein complex known as IsiA or CP43′. Long-term iron deficiency results in the formation of large IsiA aggregates, some of which associate with photosystem I (PSI) while others are not connected to a photosystem. The fluorescence at room temperature of these unconnected aggregates is strongly quenched, which points to a photoprotective function. In this study, we report time-resolved fluorescence measurements of IsiA aggregates at low temperatures. The average fluorescence lifetimes are estimated to be about 600 ps at 5 K and 150 ps at 80 K. Both lifetimes are much shorter than that of the monomeric complex CP47 at 77 K. We conclude that IsiA aggregates quench fluorescence to a significant extent at cryogenic temperatures. We show by low-temperature fluorescence spectroscopy that unconnected IsiA is present already after two days of growth in an iron-deficient medium, when PSI and PSII are still present in significant amounts and that under these conditions the fluorescence quenching is similar to that after 18 days, when PSI is almost completely absent. We conclude that unconnected IsiA provides photoprotection in all stages of iron deficiency.     

pkn22 (alr2502) encoding a putative Ser/Thr kinase in the cyanobacterium Anabaena sp. PCC 7120 is induced by both iron starvation and oxidative stress and regulates the expression of isiA

In cyanobacteria, the isiA gene is required for cell adaptation to oxidative damage caused by the absence of iron. We show here that a putative Ser/Thr kinase gene, pkn22 (alr2052), is activated by iron deficiency and oxidative damage in Anabaena sp. PCC 7120. A pkn22 insertion mutant is unable to grow when iron is limiting. pkn22 regulates the expression of isiA (encoding CP43'), but not of isiB (encoding flavodoxin) and psbC (CP43). Fluorescence measurement at 77 K reveals the absence of the typical signature of CP43' associated with photosystem I in the mutant under iron-limiting conditions. We propose that Pkn22 is required for the function of isiA/CP43' and constitutes a regulatory element necessary for stress response.     

Function,regulation and distribution of IsiA,a membrane-bound chlorophyll a-antenna protein in cyanobacteria

Photosynthetica - IsiA is a membrane-bound Chl a-antenna protein synthesized in cyanobacteria under iron deficiency. Since iron deficiency is a common nutrient stress in significant fractions of...     

Structure and functional role of supercomplexes of IsiA and Photosystem I in cyanobacterial photosynthesis

Cyanobacteria express large quantities of the iron stress-inducible protein IsiA under iron deficiency. IsiA can assemble into numerous types of single or double rings surrounding Photosystem I. These supercomplexes are functional in light-harvesting, empty IsiA rings are effective energy dissipaters. Electron microscopy studies of these supercomplexes show that Photosystem I trimers bind 18 IsiA copies in a single ring, whereas monomers may bind up to 35 copies in two rings. Work on mutants indicates that the PsaF/J and PsaL subunits facilitate the formation of closed rings around Photosystem I monomers but are not obligatory components in the formation of Photosystem I-IsiA supercomplexes.     

Novel adaptive responses revealed by transcription profiling of a Synechocystis sp. PCC 6803 ΔisiA mutant in the presence and absence of hydrogen peroxide

The isiAB genes have proven to be highly stress-responsive under a variety of environmental conditions, including iron deficiency, high salt and oxidative stress. In order to understand the function of IsiA and its importance in oxidative stress, we constructed a knock out mutant of the isiA gene and compared differential gene expression of the DeltaisiA strain in the presence and absence of H2O2. We used the full genome microarray for the cyanobacterium Synechocystis sp. PCC 6803 as previously described [Postier BL, Wang HL, Singh A, Impson L, Andrews, HL, Klahn J, Li H, Risinger G, Pesta D, Deyholos M, Galbraith DW, Sherman LA and Burnap RL (2003) BMC Genenomics 4: 23-34]. We determined that one of the main differences in DeltaisiA compared to wild-type (in the absence of peroxide) was the induction of a gene cluster (sll1693-sll1696) that encoded genes resembling pilins or general secretory proteins (Gsp). These proteins are targeted to the cytoplasmic membrane and we suggest that they may be involved in the assembly of membrane complexes, including pigment-protein complexes. The DeltaisiA strain was more resistant to H2O2 compared to the wild-type. In the presence of 1.5 mM H2O2 for 30 min, a cluster of genes that includes a peroxiredoxin was induced 7- to 8-fold and we suggest that this peroxide scavenging enzyme is responsible for the increased peroxide resistance of the DeltaisiA strain.