Energy transfer and distribution in the red alga Porphyra perforata studied using picosecond fluorescence spectroscopy

The detailed process of excitation transfer among the antenna pigments of the red alga Porphyra perforata was investigated by measuring time-resolved fluorescence emission spectra using a single-photon timing system with picosecond resolution. The fluorescence decay kinetics of intact thalli at room temperature revealed wavelength-dependent multi-component chlorophyll a fluorescence emission. Our analysis attributes the majority of chlorophyll a fluorescence to excitation originating in the antennae of PS II reaction centers and emitted with maximum intensities at 680 and 740 nm. Each of these fluorescence bands was characterized by two kinetic decay components, with lifetimes of 340-380 and 1700-2000 ps and amplitudes varying with wavelength and the photochemical state of the PS II reaction centers. In addition, a small contribution to the long-wavelength fluorescence band is proposed to arise from chlorophyll a antennae coupled to PS I. This component displays fast decay kinetics with a lifetime of approx. 150 ps. Desiccation of the thalli dramatically increases the contribution of this fast decay component.     

Sodium exclusion and potassium retention by the red marine alga,Porphyra perforata

Cells of the red marine alga, Porphyra perforata, accumulate potassium and exclude sodium, chloride, and calcium. Various metabolic inhibitors including dinitrophenol, anoxia, and p-chloromercuribenzoate partially abolish the cells' ability to retain potassium and exclude sodium. Iodoacetate induces potassium loss only in the dark; reduced sulfur compounds offer protection against the effects of p-chloromercuribenzoate; dinitrophenol stimulates respiration at concentrations which cause potassium loss and sodium gain. Following exposure to anoxia potassium accumulation and sodium extrusion take place against concentration gradients. These movements are retarded by sodium cyanide, but are stimulated by light. Sodium entry, following long exposure to 0.6 M sucrose, occurs rapidly with the concentration gradient, while potassium entry against the concentration gradient takes place slowly, and is prevented by cyanide.     

Potassium-dependent sodium extrusion by cells of Porphyra perforata,a red marine alga

Potassium-free artificial sea water causes a loss of cell potassium and a gain of cell sodium in Porphyra perforata, which is not attributable to an inhibition of respiration. On adding KCl or RbCl to such low potassium, high sodium tissues, net accumulation of potassium or rubidium takes place, accompanied by net extrusion of sodium. Rates of potassium or rubidium accumulation and sodium extrusion are proportional to the amount of KCl or RbCl added only at low concentrations. Saturation of rates is evident at KCl or RbCl concentrations above 20-30 mM, suggesting the role of an ion carrier mechanism of transport. Evidence for and against mutually dependent sodium extrusion and potassium or rubidium accumulation is discussed.     

Phosphorylation-dependent regulation of excitation energy distribution between the two photosystems in higher plants

Phosphorylation-dependent movement of the light-harvesting complex II (LHCII) between photosystem II (PSII) and photosystem I (PSI) takes place in order to balance the function of the two photosystems. Traditionally, the phosphorylatable fraction of LHCII has been considered as the functional unit of this dynamic regulation. Here, a mechanical fractionation of the thylakoid membrane of Spinacia oleracea was performed from leaves both in the phosphorylated state (low light, LL) and in the dephosphorylated state (dark, D) in order to compare the phosphorylation-dependent protein movements with the excitation changes occurring in the two photosystems upon LHCII phosphorylation. Despite the fact that several LHCII proteins migrate to stroma lamellae when LHCII is phosphorylated, no increase occurs in the 77 K fluorescence emitted from PSI in this membrane fraction. On the contrary, such an increase in fluorescence occurs in the grana margin fraction, and the functionally important mobile unit is the PSI-LHCI complex. A new model for LHCII phosphorylation driven regulation of relative PSII/PSI excitation thus emphasises an increase in PSI absorption cross-section occurring in grana margins upon LHCII phosphorylation and resulting from the movement of PSI-LHCI complexes from stroma lamellae and subsequent co-operation with the P-LHCII antenna from the grana. The grana margins probably give a flexibility for regulation of linear and cyclic electron flow in plant chloroplasts.     

Phosphorylation-dependent regulation of excitation energy distribution between the two photosystems in higher plants

Phosphorylation-dependent movement of the light-harvesting complex II (LHCII) between photosystem II (PSII) and photosystem I (PSI) takes place in order to balance the function of the two photosystems. Traditionally, the phosphorylatable fraction of LHCII has been considered as the functional unit of this dynamic regulation. Here, a mechanical fractionation of the thylakoid membrane of Spinacia oleracea was performed from leaves both in the phosphorylated state (low light, LL) and in the dephosphorylated state (dark, D) in order to compare the phosphorylation-dependent protein movements with the excitation changes occurring in the two photosystems upon LHCII phosphorylation. Despite the fact that several LHCII proteins migrate to stroma lamellae when LHCII is phosphorylated, no increase occurs in the 77 K fluorescence emitted from PSI in this membrane fraction. On the contrary, such an increase in fluorescence occurs in the grana margin fraction, and the functionally important mobile unit is the PSI-LHCI complex. A new model for LHCII phosphorylation driven regulation of relative PSII/PSI excitation thus emphasises an increase in PSI absorption cross-section occurring in grana margins upon LHCII phosphorylation and resulting from the movement of PSI-LHCI complexes from stroma lamellae and subsequent co-operation with the P-LHCII antenna from the grana. The grana margins probably give a flexibility for regulation of linear and cyclic electron flow in plant chloroplasts.     

Nitrite regulates distribution of excitation energy between the two photosystems by causing state transition.

The mechanism of distribution of absorbed excitation energy between the two photosystems in the presence of nitrite has been investigated in spinach (Spinacia oleracea L.) thylakoid membranes. Nitrite inhibited PS II activity (H(2)O --> DCPIP reaction) and enhanced PS I activity (DCPIPH(2) --> MV reaction). Nitrite decreased the F(v)/F(m) ratio measured at room temperature and increased the F(730)/F(685) ratio measured at low temperature (77 K). These results suggested that nitrite caused a decrease in the excitation energy available to PS II and transferred more energy to PS I by the mechanism of state transition. Measurement of fluorescence excitation spectra at 77 K showed that nitrite increased the absorption cross-section of PS I antenna at the expense of chlorophyll b and LHC II. Based on these observations we have suggested a role of nitrite in causing state transition.     

Low pH-induced regulation of excitation energy between the two photosystems

Earlier studies have proposed that low pH causes state transitions in spinach thylakoid membranes. Several Arabidopsis mutants (stn7 incapable in phosphorylation of LHC II, stn8 incapable in phosphorylation of PSII core proteins, stn7 stn8 double mutant and npq4 lacking PsbS and hence qE) were used to investigate the mechanisms involved in low pH induced changes in the thylakoid membrane. We propose that protonation of PsbS at low pH is involved in enhancing energy spillover to PS I.     

Physiological adaptation to a newly observed low light intensity state in intact leaves,resulting in extreme imbalance in excitation energy distribution between the two photosystems

In intact leaves, a new physiological state is obtained reversibly at low light intensity (typically 1 W / m²), in which oxygen evolution yield, monitored by the photoacoustic method, approaches zero. In this ‘low-light’ state, irradiation with far-red (λ > 700 nm) background light immediately restores the normal oxygen yield, resulting in an unusually high Emerson enhancement ratio. Quantitative analysis of the enhancement ratio and the saturation curve of enhancement by far-red light shows that in the new state, short wavelength excitation does not reach PS I reaction centers, resulting in an extreme imbalance between the two photosystems. We suggest that adaptation to the low-light state occurs through loss of excitonic interaction between antennae of PS I and their reaction-centers. It appears also that the ‘far-red’ absorbing pigments do not participate in the disconnection and remain closely attached to the reaction centers of PS I. Their number is estimated to be less than 30 per reaction center. The disconnection of the antennae from the reaction center appears to be reversed by readaptation to ‘normal’ light levels, as well as by a brief preillumination with broad band (400–600 nm) light, acting as a trigger. In the last case, the transition to high oxygen yield state is transient. The quantum requirement of this recovery process is very small (approx. 10 hv / reaction center). The adaptation times after switching from higher to lower intensities and vice versa are in the range of minutes. The fluorescence yield remains virtually constant during adaptation to the low-light state in contrast to expectations, suggesting the possibility of cyclic electron flow around PS II in this state. In a chlorophyll-b-less barley mutant, which lacks the light-harvesting $\text{chlorophyll}\text{-}\text{a}\text{b}$ protein (LHC) (and possibly the newly discovered light-harvesting $\text{chlorophyll}\text{-}\text{a}\text{b}$ protein associated with PS I (LHC-I)), the ‘low-light’ state was absent. These results are consistent with the hypothesis that these antennae complexes participate directly in the adaptation to low light intensities.     

Effects of cell wall synthesis on cell polarity in the red alga Porphyra yezoensis

Polarity is a fundamental cell property essential for differentiation, proliferation and morphogenesis in unicellular and multicellular organisms. We have recently demonstrated that phosphatidylinositol 3-kinase (PI3K) activity is required for the establishment of anterior-posterior axis, leading to asymmetrical localization of F-actin in migrating monospores of the red alga Porphyra yezoensis. We also showed that the formation of the apical-basal axis via adhesion of monospores to the substratum after the cessation of migration requires newly synthesized proteins and does not depend on PI3K activity. However, little is known about the mechanism and regulation of axis conversion during development of monospores. In this addendum, we report our investigation as to the role of the cell wall in axis conversion. Our results indicate that inhibition of cell wall synthesis prevented the development of germlings. Also, defects in the cell wall disrupted the asymmetrical distribution of F-actin and inhibited the adhesion to the substratum that is required for establishment of apical-basal axis. Hence, we conclude that the cell wall is critical for the maintenance of cell polarity in migrating cells, which is indirectly involved in axis conversion via enabling monospores to adhere to the substratum.Key words: BFA, cell polarity, cell wall, F-actin, monospores, PI3K, Porphyra yezoensisThe initial establishment of cell polarity, which is exhibited in asymmetrical cell division and directional migration, depends on asymmetrical cues that lead to reorganization of the cytoskeleton and polarized distribution of cortical proteins and membrane lipids.^1–3 For directional migration of Dictyostelium cells and leukocytes, cells in the axialized form can rapidly change their body shape along with the formation of cell polarity in response to external impulses such as cAMP and cytokines, enabling them to migrate toward the external impulse with driving and contractile forces provided by asymmetrically distributed cytoskeletal elements.^4,5 Evidence is growing that in both asymmetrical cell division and migration, intracellular compartmentalization of phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol polyphosphate phosphatases is responsible for the asymmetrical and reciprocal distributions of PI(3,4,5)P₃ and PI(4,5)P₂ on plasma membranes. This helps cells to define their polarity by organizing polarized localization of F-actin and myosin.^6–8We used the monospores of the red alga Porphyra yezoensis to elucidate the molecular mechanisms involved in the establishment of cell polarity in plants. Migration and asymmetrical cell division are both observed during the early development of monospores released from monosporangia produced at the marginal region of the thallus.^9,10 Thus, monospores are thought to be unique and useful materials for investigating polarity determination in plant cells. In the early development of monospores, there are two different cellular axes: the anterior-posterior axis during migration and the apical-basal axis in asymmetrical cell division and upward growth of a thallus. The use of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, prevented the migration of monospores because the anterior-posterior axis cannot be established. This is evidence that the PI3K activity is essential for the establishment of cell polarity and asymmetric distribution of F-actin for migration of monospores.¹⁰ Thus, the formation of the former axis requires PI3K activity and asymmetrical distribution of F-actin.¹⁰ These results are similar to those observed in Dictyostelium cells and leukocytes, suggesting that the role of PI3K-dependent F-actin asymmetry in the establishment of cell polarity might be evolutionarily conserved in migrating eukaryotic cells. However, it is still unclear whether PI(3,4,5)P₃ corresponds to D3-phosphorylated phosphatidylinositol in P. yezoensis, since this phosphoinositide has not yet been detected in any plant cell.^11,12In addition to D3-phosphorylated phosphatidylinositols, it is well known that the cell wall plays an essential role in the establishment of cell polarity, a phenomenon documented in the brown algae Fucus.^13–15 It has been demonstrated that the cell wall is required for the fixation, but not the formation, of the apical-basal axis.^13,14 In this case, polarized secretion via Golgi apparatus is needed for synthesis of the cell wall.¹⁵ In fact, we observed that monospores whose migration was inhibited by treatment with PI3K and cytoskeleton inhibitors have no cell wall,¹⁰ suggesting the importance of the cell wall in formation and/or maintenance of the cell axis in P. yezoensis.To confirm this possibility, we used Brefeldin A (BFA), a specific inhibitor of polysaccharide biosynthesis required for cell wall formation via Golgi-derived vesicle trafficking. As shown in Figure 1A, part a, the cell wall was synthesized during migration of monospores. However, when freshly released monospores were treated with BFA for 3 h, there was no cell wall synthesis in monospores with a rounded shape or in migrating monospores with a tapered shape (Fig. 1A, part b). This evidence led us to conclude that Golgi-derived vesicle trafficking is responsible for cell wall formation in these monospores. These results also indicated that the anterior-posterior axis during migration can be established without cell wall synthesis. Indeed, F-actin accumulated at the leading edge in the migrating monospores in the presence of BFA (Fig. 1A, part d).Open in a separate windowFigure 1Effect of BFA on the cell wall synthesis, F-actin localization and development of monospores. (A) Freshly released monospores were treated with (parts b–d) or without (part a) BFA (MP Biomedicals) at 20 µM for 3 h incubation. The cell wall (parts a and b) and F-actin (parts c and d) were stained with 0.01% Fluorescent Brightener 28 (Sigma) and 5 U· mL⁻¹ Alex Flour 488 phalloidin (Molecular probe), respectively. Migrating monospores are indicated by arrowheads. (Part c) Cell with a round shape, (Part d) cell with a tapered shape during migration. Upper and lower photographs in each panel show bright field and fluorescent images, respectively. Direction of migrating monospores is indicated by an arrow. Scale bars: (Parts a and b) 10 µm; (Parts c and d) 5 µm. (B) Freshly released monospores were treated with (parts b–d) or without (part a) BFA at 20 µM for 24 h incubation. The cell wall (parts a and b) and F-actin (parts c and d) were stained with 0.01% Fluorescent Brightener 28 and 5 U· mL⁻¹ Alex Flour 488 phalloidin, respectively. Migrating monospores are indicated by arrowheads. (Part c) Cell with a round shape, (Part d) cell with a tapered shape during migration. Upper and lower photographs in each panel show bright field and fluorescent images, respectively. Direction of migrating monospores is indicated by an arrow. Scale bars: (Parts a and b) 10 µm; (Parts c and d) 5 µm. (C) Dose-dependent effect of BFA on early development of monospores. Freshly released monospores were treated with an increasing concentration (2–20 µM) of BFA for 24 h, and the number of germlings was counted. Data are presented as mean ± SD (n = 3).Next, we analyzed the relationship between cell wall synthesis and development of germlings to confirm the functional significance of cell wall synthesis in the establishment of apical-basal axis. In the control medium, the cell wall was observed in 2-celled germlings 24 h after monospores release (Fig. 1B, part a), while the BFA-treated monospores still retained the migrating form in which the cell wall was not synthesized (Fig. 1B, part b) and the adhesion of monospores to the substratum and development of germlings was prevented (Fig. 1B, parts b–d). Moreover, the rate of the development of germlings from monospores decreased in a dose-dependent manner after 24 h incubation with BFA (Fig. 1C). Notably, it is significant that the polarized localization of F-actin in migrating cells was destroyed during BFA treatment (Fig. 1B, part d), suggesting the involvement of the cell wall in the maintenance of the asymmetrical distribution of F-actin in migrating monospores. Thus, the cell wall is indispensable for maintenance of anterior-posterior axis in migrating cells. Moreover, since adhering is trigger of the formation of the apical-basal axis, synthesis of cell wall could enable cells to develop further into germlings. We therefore concluded that cell wall plays a role indirectly in axis conversion during the development of monospores. Future work should be focused on the nature of the cell wall factors involved in the maintenance of cell axis and the adhesion to the substratum and how the function and expression of these factors are regulated.In summary, the establishment and maintenance of cell polarity during development of monospores is under complex regulation. Dissecting the molecular mechanisms of this regulatory system could help in further understanding the interrelation among PI3K signaling, the actin-based system and cell wall formation, which can provide new insight into the machinery regulating the establishment and maintenance of cell polarity in plants.     

Variety in excitation energy transfer processes from phycobilisomes to photosystems I and II

Photosynthesis Research - The light-harvesting antennas of oxygenic photosynthetic organisms capture light energy and transfer it to the reaction centers of their photosystems. The light-harvesting...     

Reversible effects of moderately elevated temperature on the distribution of excitation energy between the two photosystems of photosynthesis in intact avocado leaves

Initial (F_o), maximum (F_m) and steady-state (F_s) levels of modulated chlorophyll fluorescence were measured in intact avocado leaves (Persea americana Mill.) during state 1-state 2 transitions using a combination of modulated and non-modulated lights with synchronized detection. Under normal temperature conditions (20°C), transition from state 2 to state 1 was associated with a substantial increase (about 20%) in F_m and F_o whereas the F_m/F_o ratio remained constant, reflecting increased absorption cross-section of PS II. On the contrary, at moderately elevated temperature (35°C), these fluorescence changes were very limited, indicating marked inhibition of the state regulation. The fraction of light distributed to PS II () was calculated from the F_o, F_m and F_s levels for both types of leaves. In control leaves, varied from 48% (in state 2) to values as high as 58% (in state 1). In contrast, mild heat treatment resulted in values close to 50% in both states, indicating the inability of heated leaves to reach extreme state 1. The results suggested that avocado leaves under moderately elevated temperature conditions are blocked in a state close to state 2. This effect was shown to occur in a non-injurious temperature range (as shown by the preservation of the (photoacoustically monitored) oxygen evolution activity) and to be rapidly reversed upon lowering of the temperature. Thermally induced development of state 2 (independent on the light spectral quality) could possibly be a protective mechanism to avoid photodamage of the heat-labile PS II by high light intensities which usually accompany heat stress in the field.     

Changes in the distribution of light energy between the two photosystems in spinach leaves

Time courses of chlorophyll fluorescence at room temperature and fluorescence spectra at 77 K were measured to investigate the light-induced changes in the distribution of light energy between the two photosy stems in young spinach leaves. Illumination of the dark adapted leaves with primarily system II light induced typical fluorescence transients at room temperature. Fluorescence spectra at 77 K showed that the intensity of system II fluorescence at 77 K changed nearly in parallel with the fluorescence transients at room temperature within the range from M₁ to T during illumination of the leaf. Illumination of the dark adapted leaves with light I produced an increase of system II fluorescence measured at 77 K. The characteristics of the changes induced by light I or II were different, showing that these two effects are related to different mechanisms. These results suggest that the dark state in spinach leaves is state II, that light I induces a state II to I transition, while light II induces fluorescence changes that are produced by mechanisms other than state I-state II transitions.     

Induced cultivation pattern enhanced the phycoerythrin production in red alga Porphyridium purpureum

Bioprocess and Biosystems Engineering - Porphyridium purpureum is a rich source for producing phycoerythrin (PE); however, the PE content is greatly affected by culture conditions. Researchers have...     

Effects of Salinity on Primary Processes of Photosynthesis in the Red Alga Porphyra perforata

The effects of salinity on the primary processes of photosynthesis were studied in the red alga Porphyra perforata. The results show that there are at least three sites in the photosynthetic apparatus of this alga that were affected by increased salinity. The first site, photoactivation and dark-inactivation of electron flow on the reducing side of photosystem I, was completely inhibited at high salinity. The second site, electron flow on the oxidizing side (water side) of photosystem II, was inhibited as was the re-oxidation of Q in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The third site affected by high salinity was the transfer of light energy probably from pigment system II to I. High salinity reduced the amount of light energy that reached the reaction centers of photosystem II.

These effects are discussed in relation to the mechanisms available to this plant to avoid photoinhibition when it is exposed to stresses such as high light and high salinity which are conditions that are commonly found in the intertidal habitat.

     

The amino acid sequence of ferredoxin from the red alga Porphyra umbilicalis.

The amino acid sequence of the ferrodoxin of Porphyra umbilicalis was determined by the dansyl-phenyl isothiocyanate method, on peptides obtained by tryptic, chymotryptic and thermolytic digestion of the protein or its CNBr-cleavage fragments. The molecule consists of 98 residues, has an unblocked N-terminus and shows considerable similarity with other plant-type ferredoxins. It is the first reported sequence of a red-algal ferredoxin.     

Correction to: Induced cultivation pattern enhanced the phycoerythrin production in red alga Porphyridium purpureum

Bioprocess and Biosystems Engineering - The original version of the article unfortunately contained an error in Microalgae strain and culture medium section. Below is the corrected version.     

Quantification of excitation energy distribution between photosystems based on a mechanistic model of photosynthetic electron transport

Absorbed light energy is converted into excitation energy. The excitation energy is distributed to photosystems depending on the wavelength and drives photochemical reactions. A non‐destructive, mechanistic and quantitative method for estimating the fraction of the excitation energy distributed to photosystem II (f) was developed. For the f values for two simultaneously provided actinic lights (ALs) with different spectral distributions to be estimated, photochemical yields of the photosystems were measured under the ALs and were then fitted to an electron transport model assuming the balance between the electron transport rates through the photosystems. For the method to be tested using leaves with different properties in terms of the long‐term and short‐term acclimation (adjustment of photosystem stoichiometry and state transition, respectively), the f values for red and far‐red light (R and FR) were estimated in leaves grown (~1 week) under white light without and with supplemental FR and adapted (~10 min) to R without and with supplemental FR. The f values for R were clearly greater than those for FR and those of leaves grown with and adapted to supplemental FR tended to be higher than the controls. These results are consistent with previous studies and therefore support the validity of the proposed method.     

Trehalose and (iso)floridoside production under desiccation stress in red alga Porphyra umbilicalis and the genes involved in their synthesis

The marine red alga Porphyra umbilicalis has high tolerance toward various abiotic stresses. In this study, the contents of floridoside, isofloridoside, and trehalose were measured using gas chromatography mass spectrometry (GC-MS) in response to desiccation and rehydration treatments; these conditions are similar to the tidal cycles that P. umbilicalis experiences in its natural habitats. The GC-MS analysis showed that the concentration of floridoside and isofloridoside did not change in response to desiccation as expected of compatible solutes. Genes involved in the synthesis of (iso)floridoside and trehalose were identified from the recently completed Porphyra genome, including four putative trehalose-6-phosphate synthase (TPS) genes, two putative trehalose-6-phosphate phosphatase (TPP) genes, and one putative trehalose synthase/amylase (TreS) gene. Based on the phylogenetic, conserved domain, and gene expression analyses, it is suggested that the Pum4785 and Pum5014 genes are related to floridoside and isofloridoside synthesis, respectively, and that the Pum4637 gene is probably involved in trehalose synthesis. Our study verifies the occurrences of nanomolar concentrations trehalose in P. umbilicalis for the first time and identifies additional genes possibly encoding trehalose phosphate synthases.     

Isolation of porphyran-degrading marine microorganisms from the surface of red alga, Porphyra yezoensis

Marine microorganisms degrading porphyran (POR) were found on the surface of thalli of Porphyra yezoensis. Fifteen crude microorganism groups softened and liquefied the surface of agar-rich plate medium. Among these, 11 microorganism groups degraded porphyran that consisted of sulfated polysaccharide in Porphyra yezoensis. Following isolation, 7 POR-degradable microorganisms were isolated from the 11 POR-degradable microorganism groups.