Localisation of fucoxanthin chlorophyll a/c-binding polypeptides of the centric diatom Cyclotella cryptica by immuno-electron microscopy

Antisera were raised against the C termini of three fucoxanthin chlorophyll a/c-binding polypeptides, Fcp2, Fcp4, and Fcp6, of the centric diatom Cyclotella cryptica. Immunogold electron microscopy of ultrathin-sectioned cells indicated that Fcp2 and Fcp4 are present in almost the same amounts, whereas approximately 8- to 10-fold less gold label was registered for Fcp6. Immunogold electron microscopy of freeze-fracture replicas of thylakoid membranes showed that the C termini of at least Fcp2 and Fcp4 were located in the thylakoid lumen, thus demonstrating a 3-dimensional structure similar to that already described for the chlorophyll a/b-binding light-harvesting polypeptides of higher plants.     

Characterization of fcp4 and fcp12, Two Additional Genes Encoding Light Harvesting Proteins of Cyclotella cryptica (Bacillariophyceae) and Phylogenetic Analysis of this Complex Gene Family

Abstract: Two additional cDNA clones containing genes which encode fucoxanthin chlorophyll a/c binding proteins (Fcps) of the centric diatom Cyclotella cryptica have been sequenced. The first cDNA clone containing fcp12 had an insert size of 871 base pairs (bp). The open reading frame (ORF) of 693 bp corresponds to a precursor protein of 231 amino acids with a molecular weight (Mr) of 25 200. For the mature Fcp12, a protein of 196 amino acids with a Mr of 21 700 is proposed. The second cDNA clone contained the fragmentary fcp4 with an insert of 805 bp. The ORF of 492 bp corresponds to a polypeptide of 164 amino acids with a Mr of 18 050. Phylogenetic analyses revealed that the proteins Fcp1, Fcp2, Fcp3 and Fcp5 are closely related to the Fcps of other diatoms, whereas Fcp6, Fcp7 and Fcp12 share the highest homology to the Fcp of the haptophyte Isochrysis galbana and to the light inducible proteins LI818r3 and LI818 of Chlamydomonas reinhardtii and Chlamydomonas eugametos. The subunit Fcp4 revealed some homology with the red algal LH subunits LhcaR1 and LhcaR2 of Porphyridium cruentum.     

Differential circadian expression of genes fcp2 and fcp6 in Cyclotella cryptica.

The steady-state mRNA concentrations of two fcp genes encoding fucoxanthin chlorophyll a/c light-harvesting polypeptides of the centric diatom Cyclotella cryptica were investigated over a 4-day period by RNA dot-blotting experiments. Before and during the first day of the experiment, the cultures were grown under a 12-h light/12-h dark regime. On the following 3 days, the algae were kept in darkness. On the first day, the steady-state mRNA concentration of fcp2 followed a diurnal pattern, with a maximum occurring around noon, approximately 6 h after the onset of light. The gene fcp6 also had a diurnal pattern on the first day. Its maximum, however, occurred immediately after the onset of light. During the subsequent incubation period in darkness, the diurnal pattern of expression of both fcp genes continued, thus demonstrating that their steady-state mRNA concentrations oscillated in a circadian manner.     

Subunit composition and pigmentation of fucoxanthin-chlorophyll proteins in diatoms: evidence for a subunit involved in diadinoxanthin and diatoxanthin binding

Two different fucoxanthin-chlorophyll protein complexes (FCP) were purified from the centric diatom Cyclotella meneghiniana and characterized with regard to their polypeptide and pigment composition. Whereas the oligomeric FCPb complex is most probably composed of fcp5 gene products, the trimeric FCPa has subunits encoded by fcp1-3 and fcp6/7. The amount of the latter polypeptide is enhanced when FCPa is isolated from algae grown under HL conditions. This increase in Fcp6/7 polypeptides is accompanied by an increase in the pool of xanthophyll cycle pigments, diadinoxanthin and diatoxanthin, and a concomitant decrease in fucoxanthin content. In addition, the de-epoxidation ratio, i.e., the amount of diatoxanthin in relation to the pool of xanthophyll cycle pigments, is increased by a factor of 2. With regard to fluorescence yield, HL FCPa was quenched in comparison to LL FCPa. This is in accordance with the larger amount of diatoxanthin that is bound, which is supposed to act as a quencher like zeaxanthin in higher plants. Thus, we conclude that the enhanced content of diatoxanthin in FCPa plays a protective role, which is paralleled by a weakened light harvesting function due to a smaller amount of fucoxanthin.     

The structure of FCPb,a light-harvesting complex in the diatom <Emphasis Type="Italic">Cyclotella meneghiniana</Emphasis>

Diatoms possess fucoxanthin chlorophyll proteins (FCP) as light-harvesting systems. These membrane intrinsic proteins bind fucoxanthin as major carotenoid and Chl c as accessory chlorophyll. The relatively high sequence homology to higher plant light-harvesting complex II gave rise to the assumption of a similar overall structure. From centric diatoms like Cyclotella meneghiniana, however, two major FCP complexes can be isolated. FCPa, composed of Fcp2 and Fcp6 subunits, was demonstrated to be trimeric, whereas FCPb, known to contain Fcp5 polypeptides, is of higher oligomeric state. No molecular structure of either complex is available so far. Here we used electron microscopy and single particle analysis to elucidate the overall architecture of FCPb. The complexes are built from trimers as basic unit, assembling into nonameric moieties. The trimer itself is smaller, i.e. more compact than LHCII, but the main structural features are conserved.     

Association of fucoxanthin chlorophyll a/c-binding polypeptides with photosystems and phosphorylation in the centric diatom Cyclotella cryptica

Solubilization of thylakoid membranes of Cyclotella cryptica with dodecyl-beta maltoside followed by sucrose density gradient centrifugation or deriphate polyacrylamide gel electrophoresis resulted in the isolation of pigment protein complexes. These complexes were characterized by absorption and fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western immunoblotting using antisera against fucoxanthin chlorophyll a/c-binding proteins and the reaction center protein D2 of photosystem II. Sucrose density gradient centrifugation yielded four bands. Band 1 consisted of free pigments with minor amounts of fucoxanthin chlorophyll a/c-binding proteins. Bands 2, 3, and 4 represented a major fucoxanthin chlorophyll a/c-binding protein fraction, photosystem II, and photosystem I, respectively. Deriphate polyacrylamide gel electrophoresis gave rise to five bands, representing photosystem I, photosystem II, two fucoxanthin chlorophyll a/c-binding protein complexes, and a band mostly consisting of free pigments. In the Western immunoblotting experiments, the specific association of two fucoxanthin chlorophyll a/c-binding proteins, Fcp2 and Fcp4, to the photosystems could be demonstrated. In vivo experiments using antibodies against phosphothreonine residues and in vitro studies using [gamma-32P]ATP showed that fucoxanthin chlorophyll a/c binding-proteins of 22 kDa became phosphorylated.     

The Light Harvesting System of the Diatom Cyclotella cryptica. Isolation and Characterization of the Main Light Harvesting Complex and Evidence for the Existence of Minor Pigment Proteins*

The main chlorophyll a/c light harvesting complex of the diatom Cyclotella cryptica was isolated by sucrose density gradient centrifugation. It consisted of two polypeptides of Mrs 18000 and 22000. Both polypeptides and fragments thereof, obtained by formic acid treatment, were blocked at their N-ter-mini. An antiserum raised against the two subunits selectively immunolabeled the thylakoid within the chloroplasts. The subunits were nuclear encoded and could be immunoprecipitated from poly (A)⁺ RNA as precursor proteins in the Mr range of 20000 to 24000. The existence of minor chlorophyll protein complexes and their possible function in light climate adaptation processes was investigated in cells adapted to low light and high light conditions. Low light grown cells contained more fucoxanthin and less β-carotene relative to chlorophyll a than high light adapted cells. The xanthophyll cycle pigments diatoxanthin and diadinoxanthin increased five-fold relative to chlorophyll a under high light conditions. Western-immunoblotting experiments with antisera raised against several chlorophyll a/b and chlorophyll a/c antenna complexes demonstrated that, beside the dominating chlorophyll a/c light harvesting complex, minor antenna complexes might exist, which, in part, seem to react to the light climate applied.     

Investigations on gene copy number, introns and chromosomal arrangement of genes encoding the fucoxanthin chlorophyll a/c-binding proteins of the centric diatom Cyclotella cryptica

The gene arrangement, existence of introns and the number of gene copies of genes (fcps) encoding fucoxanthin chlorophyll a/c-binding proteins (Fcps) of the centric diatom Cyclotella cryptica were investigated by polymerase chain reaction (PCR), Southern blotting and denaturing gradient gel electrophoresis (DGGE) experiments. PCR-mediated amplification of the fcp genes using chromosomal DNA as template demonstrated the absence of introns within the amplified regions. Clustering of genes could not be demonstrated in these experiments. Digestion of chromosomal DNA of Cy. cryptica followed by Southern blotting and hybridization with specific fcp probes revealed minimum and maximum values of 12 and 20, respectively, for the gene copies. In addition, the DGGE technique confirmed and strengthened the results obtained from Southern blotting experiments as amplification of gene fragments from genomic DNA with different sets of specific primers revealed values of 21 and 23, for the minimum and maximum gene copy number, respectively.     

Effects of Light intensity on Photosynthetic Characteristics of Wheat Seedling

The contents of pigments and chlorophyll-protein complexes, fluorescence characteristics and electron transport rate were compared for wheat seedlings grown under different light intensities. Leaves of wheat seedlings grown under low-light intensity (2 klx) had lower chlorophyll and carotenoid contents on leaf area or fresh weight basis, a lower ratio of chlorophyll a/b, lower CPIa and CPI contents in photosynthetic membranes than those of wheat seedlings grown under high-light intensity (20 klx). However, the LHCP content in photosynthetic membranes was higher in the former. The kinetic studies of fluorescence induction showed that wheat seedlings grown under low-light intensity possessed a bigger photosynthetic unit, lower PSⅡ activity and lower efficiency of primary energy conversion than those grown under high-light intensity. Moreover. lower electron transport rate was found in the chloroplasts of the former.     

ANATOMICAL AND PHYSIOLOGICAL ACCLIMATION OF FATSIA JAPONICA LEAVES TO IRRADIANCE

Anatomical and physiological leaf characteristics and biomass production of Fatsia japonica plants were studied. Plants were grown in a growth chamber at 300 μmol m^-2 s^-1 (high light) and 50 μmol m^-2 s^-1 (low light) photosynthetic photon flux density. Plants grown under high light showed a net maximum photosynthetic rate 44% higher than plants grown under low light; the light compensation point and the light saturation point were also higher in high-light plants. Photosynthetic oxygen evolution in isolated chloroplasts was about 40% higher in high-light plants. However, chlorophyll content on a dry weight basis, on a leaf area basis, and per chloroplast was greater in plants grown under low light. Leaf thickness in high-light plants was 13% higher than in low-light plants. The number of chloroplasts was 30% higher in high-light leaves, while chloroplast size was only slightly higher. Chloroplast ultrastructure was also affected by light. Leaf dry weight, leaf area, and biomass production per plant were drastically reduced under low light. Thus, F. japonica is a plant that is able to acclimate to different photosynthetic photon flux density by altering its anatomical and physiological characteristics. However, low-light acclimation of this plant has a considerable limiting effect on biomass production.     

Identification of a specific fucoxanthin-chlorophyll protein in the light harvesting complex of photosystem I in the diatom Cyclotella meneghiniana

Thylakoids of the diatom Cyclotella meneghiniana were separated by discontinuous gradient centrifugation into photosystem (PS) I, PSII, and fucoxanthin-chlorophyll protein (FCP) fractions. FCPs are homologue to light harvesting complexes of higher plants with similar function in e.g. brown algae and diatoms. Still, it is unclear if FCP complexes are specifically associated with either PSI or PSII, or if FCP complexes function as one antenna for both photosystems. However, a trimeric FCP complex, FCPa, and a higher FCP oligomer, FCPb, have been described for C. meneghiniana, already. In this study, biochemical and spectroscopical evidences are provided that reveal a different subset of associated Fcp polypeptides within the isolated photosystem complexes. Whereas the PSII associated Fcp antenna resembles FCPa since it contains Fcp2 and Fcp6, at least three different Fcp polypeptides are associated with PSI. By re-solubilisation and a further purification step Fcp polypeptides were partially removed from PSI and both fractions were analysed again by biochemical and spectroscopical means, as well as by HPLC. Thereby a protein related to Fcp4 and a so far undescribed 17 kDa Fcp were found to be strongly coupled to PSI, whereas presumably Fcp5, a subunit of the FCPb complex, is only loosely bound to the PSI core. Thus, an association of FCPb and PSI is assumed.     

The monomeric photosystem I-complex of the diatom Phaeodactylum tricornutum binds specific fucoxanthin chlorophyll proteins (FCPs) as light-harvesting complexes

A photosystem I (PSI)-fucoxanthin chlorophyll protein (FCP) complex with a chlorophyll a/P700 ratio of approximately 200:1 was isolated from the diatom Phaeodactylum tricornutum. Spectroscopic analysis proved that the more tightly bound FCP functions as a light-harvesting complex, actively transferring light energy from its accessory pigments chlorophyll c and fucoxanthin to the PSI core. Using an antibody against all FCP polypeptides of Cyclotella cryptica it could be shown that the polypeptides of the major FCP fraction differ from the FCPs found in the PSI fraction. Since these FCPs are tightly bound to PSI, active in energy transfer, and not found in the main FCP fraction, we suppose them to be PSI specific. Blue Native-PAGE, gel filtration and first electron microscopy studies of the PSI-FCP sample revealed a monomeric complex comparable in size and shape to the PSI-LHCI complex of green algae.     

Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states

Fucoxanthin-chlorophyll proteins were purified from the centric diatom Cyclotella meneghiniana. Two major fractions were observed that differed in their polypeptide composition and oligomeric state. Trimers consist of mainly 18 kDa polypeptides. Higher oligomers are tightly assembled from different trimers, which contain mostly 19 kDa subunits. In both oligomeric states, the excitation energy coupling between fucoxanthin and chlorophyll a was preserved, and chlorophyll c was shown to transfer energy efficiently to chlorophyll a. Circular dichroism spectra showed close interaction between fucoxanthin and chlorophyll a, and different chlorophyll a molecules were demonstrated to interact excitonically. The assembly of trimers of antenna proteins with a distinct subunit composition into higher oligomeric states was not reported so far and differs from the situation found in higher plants. The differences in the supramolecular structure of the fucoxanthin-chlorophyll proteins reflect the dissimilar arrangement of the thylakoid membranes in diatoms, which lack the grana-stroma distinction.     

Improvement of acclimatization of micropropagated grapevine: Photosynthetic competence and carbon allocation

Grapevine plantlets multiplied in vitro were acclimatized at 40 or 90 μmol m⁻² s⁻¹ photon flux density for 12 or 16 h per day, respectively. In the high-light regime a decrease in total chlorophyll and an increase in chlorophyll a/chlorophyll b ratio occurred. However, at high-light intensity lower photosynthetic capacities and higher apparent photosynthesis were measured than at the low-light regime. In leaves expanded during acclimatization, the light compensation point was higher in plantlets under high-light while quantum yield was higher in low-light conditions. High-light also gave rise to an increase in carbohydrate concentration. As a whole, the results suggest that high-light increases carbon assimilation and growth although with a low investment in the photosynthetic apparatus. This revised version was published online in June 2006 with corrections to the Cover Date.     

The monomeric photosystem I-complex of the diatom Phaeodactylum tricornutum binds specific fucoxanthin chlorophyll proteins (FCPs) as light-harvesting complexes

A photosystem I (PSI)-fucoxanthin chlorophyll protein (FCP) complex with a chlorophyll a/P700 ratio of approximately 200:1 was isolated from the diatom Phaeodactylum tricornutum. Spectroscopic analysis proved that the more tightly bound FCP functions as a light-harvesting complex, actively transferring light energy from its accessory pigments chlorophyll c and fucoxanthin to the PSI core. Using an antibody against all FCP polypeptides of Cyclotella cryptica it could be shown that the polypeptides of the major FCP fraction differ from the FCPs found in the PSI fraction. Since these FCPs are tightly bound to PSI, active in energy transfer, and not found in the main FCP fraction, we suppose them to be PSI specific. Blue Native-PAGE, gel filtration and first electron microscopy studies of the PSI-FCP sample revealed a monomeric complex comparable in size and shape to the PSI-LHCI complex of green algae.     

Effects of Light and Nutrients on Leaf Size, CO(2) Exchange, and Anatomy in Wild Strawberry (Fragaria virginiana)

Plants of a single genotype of wild strawberry, Fragaria virginiana Duchesne, were grown with or without fertilizer in high (406 microeinsteins per square meter per second) and low (80 microeinsteins per square meter per second) light. High-light leaves were thicker than low-light leaves and had greater development of the mesophyll. Within a light level, high-nutrient leaves were thicker, but the proportions of leaf tissues did not change with nutrient level. Maximum net CO₂ exchange rate and leaf size were greatest in high-light, high-nutrient leaves and lowest in high-light, low-nutrient leaves. Changes in mesophyll cell volume largely accounted for differences in CO₂ exchange rate in low-light leaves, but not in high-light leaves.

Leaf size in these experiments was apparently determined by nutrient and carbon supply. This may explain the observation that the largest leaves produced by wild strawberries in the field occur in high-light, mesic habitats, rather than in shady habitats.