Submarine pollination in the marine angiosperm Zostera marina (Zosteraceae). I. The influence of floral morphology on fluid flow

An understanding of the process of submarine pollination should provide insight into the evolutionary and reproductive ecology of the marine angiosperms (seagrasses). The flow around the reproductive organs of the seagrass Zostera marina L. (Potamogetonales) was, therefore, examined in a flow chamber. The phenological emergence of flowers during (1) pollen capture and (2) pollen release, and by fruit during (3) seed release, led to a reduction in flow rate toward the inflorescence. This change in flow due to floral emergence was associated with a 50% increase in the fluid shear stress [tau = (2.2 _ 0.3) x 10-3 Pa for an immature flower vs. tau = (3.1 _ 0.5) x 10-3 Pa for a receptive flower]. The Reynolds number (Re) and fluid shear stress around inflorescences and infructescences were comparable, indicating a dynamic similarity in the processes of pollen capture and fruit dehiscence [Re = 47 _ 5, tau = (1.6 _ 0.3) x 10-3 Pa for inflorescences; Re = 38 _ 5, tau = (1.3 _ 0.1) x 10-3 Pa for infructescences]. These results indicate that the emergence of reproductive organs leads to changes in fluid shear stress, which will affect the release, transport, and capture of particles including pollen. Theoretical considerations of these observations using aerosol-filtration theory suggest that pollen capture in Z. marina occurs through direct interception of pollen by stigmas.     

Salt-tolerant ATPase activity in the plasma membrane of the marine angiosperm Zostera marina L

Plasma membrane (PM) H(+)-ATPase and H(+) transport activity were detected in PM fractions prepared from Zostera marina (a seagrass), Vallisneria gigantea (a freshwater grass) and Oryza sativa (rice, a terrestrial plant). The properties of Z. marina PM H(+)-ATPase, specifically, the optimal pH for ATPase activity and the result of trypsin treatment, were similar to those of authentic PM H(+)-ATPases in higher plants. In V. gigantea and O. sativa PM fractions, vanadate-sensitive (P-type) ATPase activities were inhibited by the addition of NaCl. In contrast, activity in the Z. marina PM fraction was not inhibited. The nitrate-sensitive (V-type) and azide-sensitive (F-type) ATPase activities in the Z. marina crude microsomal fraction and the cytoplasmic phosphoenolpyruvate carboxylase activity, however, were inhibited by NaCl, indicating that not all enzyme activities in Z. marina are insensitive to salt. Although the ratio of Na(+) to K(+) (Na(+)/K(+)) in seawater is about 30, Na(+)/K(+) in the Z. marina cells was about 1.0. The salt-tolerant ATPase activity in the plasma membrane must play an important role in maintaining a low Na(+) concentration in the seagrass cells.     

Fitness‐consequences of geitonogamous selfing in a clonal marine angiosperm (Zostera marina)

Plant mating systems have received considerable attention because the proportion of selfed vs. outcrossed progeny is an important evolutionary factor. In clonally reproducing plants, geitonogamous selfing between distant ramets belonging to the same genet is expected to be widespread, yet empirical data are sparse. Nothing is known about between‐ramet selfing in aquatic flowering plants with subaqueous pollen transfer, most of which display pronounced clonal reproduction. From two locations in the western Baltic Sea, I present data on the effects of patch isolation and clonal diversity on the outcrossing rate of eelgrass, Zostera marina L., based on the genotypes of maternal plants and recently fertilized ovules scored at eight microsatellite loci. There were no differences in outcrossing rates between vegetation patches and continuous meadow although patches were nearly always composed of single genets. Quantitative effects of clonal diversity were present in the continuous vegetation where a significant positive correlation between genet diversity and the proportion of outcrossed offspring was detected (Kendall’s τ=0.82, P=0.0017). On a population‐scale as well, the genotypic diversity was positively correlated with outcrossing. The relative fitness of selfed offspring was low (ω ± 95% confidence interval=0.56 ± 0.032 and 0.322 ± 0.15) indicating that geitonogamy incurred substantial fitness costs. Selfing rates in Z. marina may not be in evolutionary equilibrium because of spatial and temporal heterogeneity of clonal size and diversity. The high prevalence of dioecy in seagrasses may have evolved to avoid the fitness costs associated with geitonogamy.     

Submarine pollination in the marine angiosperm Zostera marina (Zosteraceae). II. Pollen transport in flow fields and capture by stigmas

Flow chamber observations of the filamentous pollen of Zostera marina L. (Potamogetonales) revealed that pollen rotated and moved toward inflorescences where they were captured by stigmas. The mechanics of this abiotic pollination process were examined and found to be related to the flow environment around emergent flowers. The translational movement of pollen was imparted by the advection of the fluid (e.g., pollen kinetic energy, K, ranged from 0.8 x 10-14 to 2.4 x 10-14 J, and the average K of the fluid was _ 0.7 x 10-14 J), while the rotational motion was imparted by the fluid shear stress (tau) within the velocity gradient (e.g., pollen shear stress, sigmat = omegamu where omega is the rotational velocity and mu is the dynamic viscosity, ranged from 3.4 x 10-4 to 26 x 10-4 Pa, and the average fluid shear stress was tau _ 10 x 10-4 Pa; Ackerman, 1997, American Journal of Botany 84: 1099-1109). These results indicate that there is a greater potential for pollination by filamentous pollen relative to spherical pollen. Functionally, while spherical pollen needs to be directly upstream from stigmas to be captured, filamentous pollen need only be in the vicinity of inflorescences and flowers to be captured by stigmas. Thus, in addition to direct interception on stigmas, filamentous pollen can be captured while they rotate past flowers or when they are redirected through the velocity gradient towards flowers. Filamentous pollen is an adaptation to submarine pollination in seagrasses.     

Genome scans detect consistent divergent selection among subtidal vs. intertidal populations of the marine angiosperm Zostera marina

Genome scans are a powerful tool to detect natural selection in natural populations among a larger sample of marker loci. We used replicated habitat comparisons to search for consistent signals of selection among contrasting populations of the seagrass Zostera marina, a marine flowering plant with important ecological functions. We compared two different habitat types in the North Frisian Wadden Sea, either permanently submerged (subtidal) or subjected to aerial exposure (intertidal). In three independent population pairs, each consisting of one tidal creek and one tidal flat population each, we carried out a genome scan with 14 expressed sequence tag (EST)-derived microsatellites situated in 5'- or 3'-untranslated regions of putative genes, in addition to 11 anonymous genomic microsatellites. By using two approaches for outlier identification, one anonymous and two EST-derived microsatellites showed population differentiation patterns not consistent with neutrality. These microsatellites were detected in several parallel population comparisons, suggesting that they are under diverging selection. One of these loci is linked to a putative nodulin gene, which is responsible for water channelling across cellular membranes, suggesting a functional link of the observed genetic divergence with habitat characteristics.     

The relationship between state II to state I transitions and cyclic electron flow around photosystem I

The effects of electron acceptors, inhibitors of electron flow and uncouplers and inhibitors of photophosphorylation on a state II to I transition were studied. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not inhibit the state II to I transition. By contrast, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), methyl viologen and antimycin A inhibited the transition indicating that the cyclic electron flow around photosystem I, but not the oxidation of electron carriers (such as plastoquinone), induced the state II to I transition. Uncouplers, but not inhibitors of photophosphorylation, inhibited the state transition suggesting that the proton transport through the cyclic electron flow was related to the transition.     

The relationship between state II to state I transitions and cyclic electron flow around photosystem I

The effects of electron acceptors, inhibitors of electron flow and uncouplers and inhibitors of photophosphorylation on a state II to I transition were studied. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not inhibit the state II to I transition. By contrast, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), methyl viologen and antimycin A inhibited the transition indicating that the cyclic electron flow around photosystem I, but not the oxidation of electron carriers (such as plastoquinone), induced the state II to I transition. Uncouplers, but not inhibitors of photophosphorylation, inhibited the state transition suggesting that the proton transport through the cyclic electron flow was related to the transition.     

Redox modulation of cyclic electron flow around photosystem I in C3 plants

We have investigated the occurrence of cyclic electron flow in intact spinach leaves. In particular, we have tested the hypothesis that cyclic flow requires the presence of supercomplexes in the thylakoid membrane or other strong associations between proteins. Using biochemical approaches, we found no evidence of the presence of supercomplexes related to cyclic electron flow, making previous structural explanations for the modulation of cyclic flow rather unlikely. On the other hand, we found that the fraction of photosystem I complexes engaged in cyclic flow could be modulated by changes in the redox state of the chloroplast stroma. Our findings support therefore a dynamic model for the occurrence of linear and cyclic electron flow in C3 plants, based on the competition between cytochrome b(6)f and FNR for electrons carried by ferredoxin. This would be ultimately regulated by the balance between the redox state of PSI acceptors and donors during photosynthesis, in a diffusing system.     

Contribution of NDH-dependent cyclic electron transport around photosystem I to the generation of proton motive force in the weak mutant allele of pgr5

In angiosperms, cyclic electron transport (CET) around photosystem I (PSI) consists of two pathways, depending on PGR5/PGRL1 proteins and the chloroplast NDH complex. In single mutants defective in chloroplast NDH, photosynthetic electron transport is only slightly affected at low light intensity, but in double mutants impaired in both CET pathways photosynthesis and plant growth are severely affected. The question is whether this strong mutant phenotype observed in double mutants can be simply explained by the additive effect of defects in both CET pathways. In this study, we used the weak mutant allele of pgr5-2 for the background of double mutants to avoid possible problems caused by the secondary effects due to the strong mutant phenotype. In two double mutants, crr2-2 pgr5-2 and ndhs-1 pgr5-2, the plant growth was unaffected and linear electron transport was only slightly affected. However, NPQ induction was more severely impaired in the double mutants than in the pgr5-2 single mutant. A similar trend was observed in the size of the proton motive force. Despite the slight reduction in photosystem II parameters, PSI parameters were severely affected in the pgr5-2 single mutant, the phenotype that was further enhanced by adding the NDH defects. Despite the lack of ?pH-dependent regulation at the cytochrome b₆f complex (donor-side regulation of PSI), the plastoquinone pool was more reduced in the double mutants than in the pgr5-2 single mutants. This phenotype suggests that both PGR5/PGRL1- and NDH-dependent CET contribute to supply sufficient acceptors from PSI by balancing the ATP/NADPH production ratio.     

Studies on photosystem I. II. Involvement of ferredoxin in cyclic electron flow

The effects of ferredoxin (Fd) and ferredoxin-NADP reductase on the light-induced spectral changes of cytochrome f (cyt f) were investigated with specific reference to their possible involvement in the cyclic electron transfort pathway of photosystem I (PS I). The steady-state level of photooxidation of reduced cytochrome f is decreased by ferredoxin but unaffected by either ferredoxin-NADP reductase alone or ferredoxin plus ferredoxin-NADP reductase when present in equimolar concentrations. These data are taken as evidence for a cyclic electron transport pathway of: PS I → “X” → Fd → (cyt f) → PC → PS I. The reduced ferredoxin could either reduce directly plastocyanin (PC) or via cytochrome f; the data do not allow differentiation between these two possibilities. However, neither ferredoxin-NADP reductase nor cytochrome b564 appear to serve as electron carriers in this pathway.     

Physiological evidence for a proton pump and sodium exclusion mechanisms at the plasma membrane of the marine angiosperm Zostera marina L

The basic electrical plasma membrane characteristics of leaf cells from the seagrass Zostera marina L. have been investigated with respect to its primary transport system and its Na⁺/K⁺ selectivity. In natural seawater Z. marina exhibits a membrane potential of -15610 mV. The phytotoxin fusicoccin stimulates H⁺ extrusion and hyperpolarizes the plasma membrane. Ouabain, an inhibitor of the mammalian Na⁺K⁺-ATPase did not depolarize the plasma membrane of Z.marina. Both flushing the leaves with CO2 and 'light off' acidified the cytoplasm and hyperpolarized the cells. It is suggested that a H⁺-ATPase rather than a Na⁺-ATPase is the primary pump in Z.marina. In the presence of cyanide plus salicylhydroxamic acid the membrane potential changed to -6411 mV. This so-called diffusion potential was sensitive to external [K⁺] from 0.05 to 0.5 mM in the presence of 0.5 M Na⁺ and revealed a relative permeability PK⁺/PNa⁺ of 303. We suggest that this high ratio is the basic adaptation which permits Z. marina to grow in high [Na⁺] conditions and to exhibit a rather negative resting potential. Since amiloride, an inhibitor of the nH⁺/Na⁺ antiporter, hyperpolarized the plasma membrane, it is suggested that this transporter could be present in the plasma membrane of Z. marina acting as an overflow valve for Na⁺ which leaks into the cell.     

Photosynthetic electron flow during leaf senescence: Evidence for a preferential maintenance of photosystem I activity and increased cyclic electron flow

Limitations in photosystem function and photosynthetic electron flow were investigated during leaf senescence in two field-grown plants, i.e., Euphorbia dendroides L. and Morus alba L., a summer- and winter-deciduous, shrub and tree, respectively. Analysis of fast chlorophyll (Chl) a fluorescence transients and post-illumination fluorescence yield increase were used to assess photosynthetic properties at various stages of senescence, the latter judged from the extent of Chl loss. In both plants, the yield of primary photochemistry of PSII and the content of PSI remained quite stable up to the last stages of senescence, when leaves were almost yellow. However, the potential for linear electron flow along PSII was limited much earlier, especially in E. dendroides, by an apparent inactivation of the oxygen-evolving complex and a lower efficiency of electron transfer to intermediate carriers. On the contrary, the corresponding efficiency of electron transfer from intermediate carriers to final acceptors of PSI was increased. In addition, cyclic electron flow around PSI was accelerated with the progress of senescence in E. dendroides, while a corresponding trend in M. alba was not statistically significant. However, there was no decrease in PSI activity even at the last stages of senescence. We argue that a switch to cyclic electron flow around PSI during leaf senescence may have the dual role of replenishing the ATP and maintaining a satisfactory nonphotochemical energy quenching, since both are limited by hindered linear electron transfer.     

Photo-oxidation of P740, the primary electron donor in photosystem I from Acaryochloris marina

Fourier transform infrared spectroscopy (FTIR) difference spectroscopy in combination with deuterium exchange experiments has been used to study the photo-oxidation of P740, the primary electron donor in photosystem I from Acaryochloris marina. Comparison of (P740(+)-P740) and (P700(+)-P700) FTIR difference spectra show that P700 and P740 share many structural similarities. However, there are several distinct differences also: 1), The (P740(+)-P740) FTIR difference spectrum is significantly altered upon proton exchange, considerably more so than the (P700(+)-P700) FTIR difference spectrum. The P740 binding pocket is therefore more accessible than the P700 binding pocket. 2), Broad, "dimer" absorption bands are observed for both P700(+) and P740(+). These bands differ significantly in substructure, however, suggesting differences in the electronic organization of P700(+) and P740(+). 3), Bands are observed at 2727(-) and 2715(-) cm(-1) in the (P740(+)-P740) FTIR difference spectrum, but are absent in the (P700(+)-P700) FTIR difference spectrum. These bands are due to formyl CH modes of chlorophyll d. Therefore, P740 consists of two chlorophyll d molecules. Deuterium-induced modification of the (P740(+)-P740) FTIR difference spectrum indicates that only the highest frequency 13(3) ester carbonyl mode of P740 downshifts, indicating that this ester mode is weakly H-bonded. In contrast, the highest frequency ester carbonyl mode of P700 is free from H-bonding. Deuterium-induced changes in (P740(+)-P740) FTIR difference spectrum could also indicate that one of the chlorophyll d 3(1) carbonyls of P740 is hydrogen bonded.     

Cyclic electron flow around photosystem I in unicellular green algae

Cyclic electron flow around PSI, or cyclic photophosphorylation, is the photosynthetic process which recycles the reducing equivalents produced by photosystem I in the stroma towards the plastoquinone pool. Through the activity of cytochrome b ₆ f, which also transfers protons across the membrane, it promotes the synthesis of ATP. The literature dealing with cyclic electron flow in unicellular algae is far less abundant than it is for plants. However, in the chloroplast of algae such as Chlorella or Chlamydomonas, an efficient carbohydrate catabolism renders the redox poise much more reducing than in plant chloroplasts. It is therefore worthwhile highlighting the specific properties of unicellular algae because cyclic electron flow is highly dependent upon the accumulation of these stromal reducing equivalents. Such an increase of reducing power in the stroma stimulates the reduction of plastoquinones, which is the limiting step of cyclic electron flow. In anaerobic conditions in the dark, this reaction can lead to a fully reduced plastoquinone pool and induce state transitions, the migration of 80% of light harvesting complexes II and 20% of cytochrome b ₆ f complex from the PSII-enriched grana to the PSI-enriched lamella. These ultrastructural changes have been proposed to further enhance cyclic electron flow by increasing PSI antenna size, and forming PSI-cyt b ₆ f supercomplexes. These hypotheses are discussed in light of recently published data.     

Characterization of highly purified photosystem I complexes from the chlorophyll d-dominated cyanobacterium Acaryochloris marina MBIC 11017

Photochemically active photosystem (PS) I complexes were purified from the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina MBIC 11017, and several of their properties were characterized. PS I complexes consist of 11 subunits, including PsaK1 and PsaK2; a new small subunit was identified and named Psa27. The new subunit might replace the function of PsaI that is absent in A. marina. The amounts of pigments per one molecule of Chl d' were 97.0 +/- 11.0 Chl d, 1.9 +/- 0.5 Chl a, 25.2 +/- 2.4 alpha-carotene, and two phylloquinone molecules. The light-induced Fourier transform infrared difference spectroscopy and light-induced difference absorption spectra reconfirmed that the primary electron donor of PS I (P740) was the Chl d dimer. In addition to P740, the difference spectrum contained an additional band at 728 nm. The redox potentials of P740 were estimated to be 439 mV by spectroelectrochemistry; this value was comparable with the potential of P700 in other cyanobacteria and higher plants. This suggests that the overall energetics of the PS I reaction were adjusted to the electron acceptor side to utilize the lower light energy gained by P740. The distribution of charge in P740 was estimated by a density functional theory calculation, and a partial localization of charge was predicted to P1 Chl (special pair Chl on PsaA). Based on differences in the protein matrix and optical properties of P740, construction of the PS I core in A. marina was discussed.     

An electron paramagnetic resonance investigation of the electron transfer reactions in the chlorophyll d containing photosystem I of Acaryochloris marina

Electron paramagnetic resonance (EPR) spectroscopy reveals functional and structural similarities between the reaction centres of the chlorophyll d-binding photosystem I (PS I) and chlorophyll a-binding PS I. Continuous wave EPR spectrometry at 12K identifies iron-sulphur centres as terminal electron acceptors of chlorophyll d-binding PS I. A transient light-induced electron spin echo (ESE) signal indicates the presence of a quinone as the secondary electron acceptor (Q) between P(740)(+) and the iron-sulphur centres. The distance between P(740)(+) and Q(-) was estimated within point-dipole approximation as 25.23+/-0.05A, by the analysis of the electron spin echo envelope modulation.