Nuclear localization of a hypoxia-inducible novel non-symbiotic hemoglobin in cultured alfalfa cells.

We have isolated a 483-bp-long full-length cDNA clone encoding a non-symbiotic hemoglobin called Mhb1, the first one found in alfalfa. This non-symbiotic hemoglobin is a single copy gene localized in linkage group 4 in diploid Medicago genome. The Mhb1 mRNA was found only in the roots of alfalfa plants. The Mhb1 gene was inducible by hypoxia and showed no induction by cold stress treatment. The Mhb1 transcript level increased at the G2/M boundary in a synchronized alfalfa cell suspension culture. The majority of Mhb1 protein was shown to be localized in the nucleus and smaller amounts were detected in the cytoplasm. A potential link to the nitric oxide signalling pathway is also discussed.

Self-assembly of large, ordered lamellae from non-bilayer lipids and integral membrane proteins in vitro.

In many biological membranes, the major lipids are "non-bilayer lipids," which in purified form cannot be arranged in a lamellar structure. The structural and functional roles of these lipids are poorly understood. This work demonstrates that the in vitro association of the two main components of a membrane, the non-bilayer lipid monogalactosyldiacylglycerol (MGDG) and the chlorophyll-a/b light-harvesting antenna protein of photosystem II (LHCII) of pea thylakoids, leads to the formation of large, ordered lamellar structures: (i) thin-section electron microscopy and circular dichroism spectroscopy reveal that the addition of MGDG induces the transformation of isolated, disordered macroaggregates of LHCII into stacked lamellar aggregates with a long-range chiral order of the complexes; (ii) small-angle x-ray scattering discloses that LHCII perturbs the structure of the pure lipid and destroys the inverted hexagonal phase; and (iii) an analysis of electron micrographs of negatively stained 2D crystals indicates that in MGDG-LHCII the complexes are found in an ordered macroarray. It is proposed that, by limiting the space available for MGDG in the macroaggregate, LHCII inhibits formation of the inverted hexagonal phase of lipids; in thylakoids, a spatial limitation is likely to be imposed by the high concentration of membrane-associated proteins.

Role of thylakoid lipids in the structural flexibility of lamellar aggregates of the isolated light-harvesting chlorophyll a/b complex of photosystem II.

We studied the role of added thylakoid lipids in the light-induced reversible structural changes in isolated macroaggregates of the main light-harvesting chlorophyll a/b complex of photosystem II (LHCII). Loosely stacked lamellar macroaggregates were earlier shown to undergo light-induced reversible structural changes and changes in the photophysical pathways, which resembled those in thylakoid membranes exposed to excess light [Barzda, V., et al. (1996) Biochemistry 35, 8981-8985]. This structural flexibility of LHCII depends critically on the lipid content of the preparations [Simidjiev, I., et al. (1997) Anal. Biochem. 250, 169-175]. It is now reported that lamellar aggregates of LHCII are capable of incorporating substantial amounts of different thylakoid lipids. The long-range order of the chromophores is retained, while the ultrastructure of the lipid-protein macroaggregates can be modified significantly. Addition of thylakoid lipids to the preparations significantly enhances the ability of the LHCII macroaggregates to undergo light-induced structural changes. The lipid environment of the LHCII complexes therefore plays a significant role in determining the structural flexibility of the macroaggregates. As concerns the mechanism of these changes, it is proposed that the absorption of light and the dissipation of its energy in the macrodomains induces thermal fluctuations which bring about changes in the shape or in the stacking interactions of the membranes, this in turn affecting the long-range order of the embedded chromophores. In thylakoids, a similar mechanism is likely to explain the light-induced structural changes which are largely independent of the photochemical activity of the membranes.

Isolation of lamellar aggregates of the light-harvesting chlorophyll a/b protein complex of photosystem II with long-range chiral order and structural flexibility.

Isolation of LHCII, the light-harvesting chlorophyll a/b complex of photosystem II, based on the procedure described by Krupa et al. (1987, Plant Physiol. 84, 19-24), was optimized for obtaining purified lamellar aggregates with long-range chiral order and structural flexibility (the capability of undergoing light-induced reversible structural changes). By varying the concentration of the detergent Triton X-100 for the solubilization of thylakoid membranes, we obtained four types of LHCII aggregates: (i) With low detergent concentration, < or = 0.6% (v/v), the aggregates contained lipids in high amount. These preparations with Chl a/b ratios of about 1.4 contained minor antenna complexes with a fingerprint of an additional CD band at (+) 505 nm; they formed disordered lamellae and exhibited no or weak psi-type CD bands (psi, polymerization- or salt-induced), which did not possess the ability to undergo light-induced changes (deltaCD). (ii) At the optimal concentration, around 0.7 +/- 0.1% (v/v), the detergent removed some lipids and most of the minor complexes, and the Chl a/b ratio dropped to 1.0-1.1. LHCII formed loosely stacked two-dimensional lamellae which exhibited psi-type CD bands and large light-induced reversible structural changes (deltaCD). (iii) At detergent concentration above the optimum, around 0.8-1% (v/v), the lipid content of LHCII decreased and minor complexes could not be detected. LHCII formed disordered aggregates and showed neither psi-type CD nor deltaCD. (iv) High concentrations (> or = 1.1% (v/v)) Triton X-100 led to very pure but largely delipidated samples assembled into tightly stacked three-dimensional lamellar structures with intense psi-type CD but no deltaCD.

Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress.

Glycinebetaine is one of the compatible solutes that accumulate in the chloroplasts of contain halotolerant plants when these plants are exposed to salt or cold stress. The codA gene for choline oxidase, the enzyme that converts choline into glycinebetaine, has previously been cloned from a soil bacterium, Arthrobacter globiformis. Transformation of Arabidopsis thaliana with the cloned codA gene under the control of the 35S promoter of cauliflower mosaic virus enabled the plant to accumulate glycinebetaine and enhanced its tolerance to salt and cold stress. At 300 mM NaCl, considerable proportions of seeds of transformed plants germinated well, whereas seeds of wild-type plants failed to germinate. At 100 mM NaCl, transformed plants grew well whereas wild-type plants did not do so. The transformed plants tolerated 200 mM NaCl, which was lethal to wild-type plants. After plants had been incubated with 400 mM NaCl for two days, the photosystem II activity of wild-type plants had almost completely disappeared, whereas that of transformed plants remained at more than 50% of the original level. When exposed to a low temperature in the light, leaves of wild-type plants exhibited symptoms of chlorosis, whereas those of transformed plants did not. These observations demonstrate that the genetic modification of Arabidopsis thaliana that allowed it to accumulate glycinebetaine enhanced its ability to tolerate salt and cold stress.

Effects of the application of aluminium, strontium and magnesium solutions to the leaf surface of maize, and determinations of these elements by ICP-AES.

The uptakes of aluminium, magnesium and strontium through maize leaves were investigated. The plants were grown in the light and in the dark (etiolation). Samples of leaves were digested with a mixture of nitric acid and hydrogen peroxide, and measurements were made by ICP-AES. The aluminium, magnesium and strontium contents of control samples grown in the light or in the dark were found to be similar. There were significant increases in the contents of these elements after maize leaves were sprayed with aluminium, magnesium or strontium solutions: increases of 100 per cent for aluminium, 20 per cent for magnesium and 10 per cent for strontium. The uptakes of these elements were generally higher in the light than in the dark. Aluminium, magnesium and strontium ions act as antagonists of each other. The effects of spraying with the different solutions were also observed in the colour of the maize leaves. However, the ultrastructure of the leaves did not change. The formation of chlorophyll was accelerated by magnesium, and inhibited by aluminium or strontium.

Immunocytochemical localization of acyl-lipid desaturases in cyanobacterial cells: evidence that both thylakoid membranes and cytoplasmic membranes are sites of lipid desaturation.

There are four acyl-lipid desaturases in the cyanobacterium Synechocystis sp. PCC 6803. Each of these desaturases introduces a double bond at a specific position, such as the Delta6, Delta9, Delta12, or omicron3 position, in C18 fatty acids. The localization of the desaturases in cyanobacterial cells was examined immunocytochemically with antibodies raised against synthetic oligopeptides that corresponded to the carboxyl-terminal regions of the desaturases. All four desaturases appeared to be located in the regions of both the cytoplasmic and the thylakoid membranes. These findings suggest that fatty acid desaturation of membrane lipids takes place in the thylakoid membranes as well as in the cytoplasmic membranes.

Transformation of Synechococcus with a gene for choline oxidase enhances tolerance to salt stress.

Choline oxidase, isolated from the soil bacterium Arthrobacter globiformis, converts choline to glycinebetaine (N-trimethylglycine) without a requirement for any cofactors. The gene for this enzyme, designated codA, was cloned and introduced into the cyanobacterium Synechococcus sp. PCC 7942. The codA gene was expressed under the control of a strong constitutive promoter, and the transformed cells accumulated glycinebetaine at intracellular levels of 60-80 mM. Consequently the cells acquired tolerance to salt stress, as evaluated in terms of growth, accumulation of chlorophyll and photosynthetic activity.

Size dependency of circular dichroism in macroaggregates of photosynthetic pigment-protein complexes.

Large molecular aggregates, condensed biological macromolecules, intact membrane systems, and cell organelles often exhibit intense anomalous circular dichroism (CD) bands which are absent in systems of lower structural complexity. Theory predicts that in dense, chirally organized macroaggregates the size of the aggregate controls the magnitude of the anomalous CD bands [Keller, D., & Bustamante, C. (1986) J. Chem. Phys. 84, 2972-2979]. Photosynthetic pigment-protein complexes in their native thylakoid membranes and in vitro were used to provide direct experimental evidence of the size dependency of CD in macroaggregates.

Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize.

A reproducible and efficient transformation system has been developed for maize that is based on direct DNA uptake into embryogenic protoplasts and regeneration of fertile plants from protoplast-derived transgenic callus tissues. Plasmid DNA, containing the beta-glucuronidase (GUS) gene, under the control of the doubled enhancer element (the -208 to -46 bp upstream fragment) from CaMV 35S promoter, linked to the truncated (up to -389 bp from ATG) promoter of wheat, alpha-amylase gene was introduced into protoplasts from suspension culture of HE/89 genotype. The constructed transformation vectors carried either the neomycin phosphotransferase (NPTII) or phosphinothricin acetyltransferase (PAT) gene as selective marker. The applied DNA uptake protocol has resulted at least in 10-20 resistant calli, or GUS-expressing colonies after treatment of 10(6) protoplasts. Vital GUS staining of microcalli has made possible the shoot regeneration from the GUS-stained tissues. 80-90% of kanamycin or PPT resistant calli showed GUS activity, and transgenic plants were regenerated from more than 140 clones. Both Southern hybridization and PCR analysis showed the presence of introduced foreign genes in the genomic DNA of the transformants. The chimeric promoter, composed of a tissue specific monocot promoter, and the viral enhancer element specified similar expression pattern in maize plants, as it was determined by the full CaMV 35S promoter in dicot and other monocot plants. The highest GUS specific activity was found in older leaves with progressively less activity in young leaves, stem and root. Histochemical localization of GUS revealed promoter function in leaf epidermis, mesophyll and vascular bundles, in the cortex and vascular cylinder of the root. In roots, the meristematic tip region and vascular tissues stained intensively. Selected transformants were grown up to maturity, and second-generation seedlings with segregation for GUS activity were obtained after outcrossing. The GUS-expressing segregants carried also the NPTII gene as shown by Southern hybridization.

Photosynthetic membrane topography: quantitative in situ localization of photosystems I and II.

An immunolabeling approach was developed for quantitative in situ labeling of photosystems I and II (PSI and PSII). Photosynthetic membranes from the phycobilisome-containing red alga Porphyridium cruentum were isolated from cells in which different photosystem compositions were predetermined by growing cells in green light (GL) or red light (RL). Based on phycobilisome densities per membrane area of 390 per m2 (GL) and 450 per m2 (RL) and the PSI reaction center (P700) and PSII reaction center (QA) content, the photosystem densities per m2 of membrane were calculated to be 2520 PSI in GL and 1580 in RL and 630 PSII in GL and 1890 in RL. PSI was detected in the membranes with 10-nm Au particles conjugated to affinity-purified anti-PSI, and PSII was detected with 15-nm Au particles conjugated to anti-PSII. Distribution of Au particles appeared relatively uniform, and the degree of labeling was consistent with the calculated photosystem densities. However, the absolute numbers of Au-labeled sites were lower than would be obtained if all reaction center monomers were labeled. Specific labeling of PSI was 25% in GL and RL membranes, and PSII labeling was 33% in GL but only 17% in RL membranes. An IgG-Au particle is larger than a monomer of either photosystem and could shield several closely packed photosystems. We suggest that clustering of photosystems exists and that the cluster size of PSI is the same in GL and RL cells, but the PSII cluster size is 2 times greater in RL than in GL cells. Such variations may reflect changes in functional domains whereby increased clustering can maximize the cooperativity between the photosystems, resulting in enhancement of the quantum yield.

Localization and quantitation of chloroplast enzymes and light-harvesting components using immunocytochemical methods.

Seven chloroplast proteins were localized in Porphyridium cruentum (ATCC 50161) by immunolabeling with colloidal gold on electron microscope sections of log phase cells grown under red, green, and white light. Ribulose bisphosphate carboxylase labeling occurred almost exclusively in the pyrenoid. The major apoproteins of photosystem I (56-64 kD) occurred mostly over the stromal thylakoid region and also appeared over the thylakoids passing through the pyrenoid. Labeling for photosystem II core components (D2 and a 45 kD Chl-binding protein), for phycobilisomes (allophycocyanin, and a 91 kD L(cm) linker) and for ATP synthase (beta subunit) were predominantly present in the thylakoid region but not in the pyrenoid region of the chloroplast. Red light cells had increased labeling per thylakoid length for polypeptides of photosystem II and of phycobilisomes, while photosystem I density decreased, compared to white light cells. Conversely, green light cells had a decreased density of photosystem II and phycobilisome polypeptides, while photosystem I density changed little compared with white light cells. A comparison of the immunogold labeling results with data from spectroscopic methods and from rocket immunoelectrophoresis indicates that it can provide a quantitative measure of the relative amounts of protein components as well as their localization in specific organellar compartments.

Stoichiometry of Photosystem I, Photosystem II, and Phycobilisomes in the Red Alga Porphyridium cruentum as a Function of Growth Irradiance.

Cells of the red alga Porphyridium cruentum (ATCC 50161) exposed to increasing growth irradiance exhibited up to a three-fold reduction in photosystems I and II (PSI and PSII) and phycobilisomes but little change in the relative numbers of these components. Batch cultures of P. cruentum were grown under four photon flux densities of continuous white light; 6 (low light, LL), 35 (medium light, ML), 180 (high light, HL), and 280 (very high light, VHL) microeinsteins per square meter per second and sampled in the exponential phase of growth. Ratios of PSII to PSI ranged between 0.43 and 0.54. About three PSII centers per phycobilisome were found, regardless of growth irradiance. The phycoerythrin content of phycobilisomes decreased by about 25% for HL and VHL compared to LL and ML cultures. The unit sizes of PSI (chlorophyll/P(700)) and PSII (chlorophyll/Q(A)) decreased by about 20% with increase in photon flux density from 6 to 280 microeinsteins per square meter per second. A threefold reduction in cell content of chlorophyll at the higher photon flux densities was accompanied by a twofold reduction in beta-carotene, and a drastic reduction in thylakoid membrane area. Cell content of zeaxanthin, the major carotenoid in P. cruentum, did not vary with growth irradiance, suggesting a role other than light-harvesting. HL cultures had a growth rate twice that of ML, eight times that of LL, and slightly greater than that of VHL cultures. Cell volume increased threefold from LL to VHL, but volume of the single chloroplast did not change. From this study it is evident that a relatively fixed stoichiometry of PSI, PSII, and phycobilisomes is maintained in the photosynthetic apparatus of this red alga over a wide range of growth irradiance.

Respiratory control over photosynthetic electron transport in chloroplasts of higher-plant cells: evidence for chlororespiration.

Flash-induced primary charge separation, detected as electrochromic absorbance change, the operation of the cytochrome b/f complex and the redox state of the plastoquinone pool were measured in leaves, protoplasts and open-cell preparations of tobacco (Nicotiana tabacum L.), and in isolated intact chloroplasts of peas (Pisum sativum L.). Addition of 0.5-5 mM KCN to these samples resulted in a large increase in the slow electrochromic rise originating from the electrogenic activity of the cytochrome b/f complex. The enhancement was also demonstrated by monitoring the absorbance transients of cytochrome f and b 6 between 540 and 572 nm. In isolated, intact chloroplasts with an inhibited photosystem (PS) II, low concentrations of dithionite or ascorbate rendered turnover of only 60% of the PSI reaction centers, KCN being required to reactivate the remainder. "Silent" PSI reaction centers which could be reactivated by KCN were shown to occur in protoplasts both in the absence and presence of a PSII inhibitor. Contrasting spectroscopic data obtained for chloroplasts before and after isolation indicated the existence of a continuous supply of reducing equivalents from the cytosol.Our data indicate that: (i) A respiratory electron-transport pathway involving a cyanide-sensitive component is located in chloroplasts and competes with photosynthetic electron transport for reducing equivalents from the plastoquinone pool. This chlororespiratory pathway appears to be similar to that found in photosynthetic prokaryotes and green algae. (ii) There is an influx of reducing equivalents from the cytosol to the plastoquinone pool. These may be indicative of a complex respiratory control of photosynthetic electron transport in higher-plant cells.

Energization and ultrastructural pattern of thylakoids formed under periodic illumination followed by continuous light.

Bean leaves grown under periodic illumination (56 cycles of 2 min light and 98 min darkness) were subsequently exposed to continuous illumination, and in connection with granum formation and accumulation of the light-harvesting pigment-protein complex thermoluminescence and light-induced shrinkage of thylakoid membranes were studied. Juvenile chloroplasts with large double sheets of thylakoids obtained under periodic light exhibited low temperature spectra of polarized fluorescence yielding fluorescence polarization (FP) values < 1 at 695 nm, characteristic for pheophytin emission. In the course of maturation under continuous light when normal grana appeared and the chlorophyll a/b light-harvesting photosystem II complex was incorporated into the membrane, at 695 nm the relative intensity of fluorescence dropped and FP changed to a value of > 1, suggesting an overlap between the emission of pheophytin and that of the chlorophyll a/b light-harvesting photosystem II complex. Thermoluminescence glow curves recorded with juvenile thylakoids displayed a relatively high proportion of emission at low temperatures (around -10°C) while with mature chloroplasts, more thermoluminescence originated from energetically deeper traps (discharged around 28°C). This means that during thylakoid development the capacity of the membrane to stabilize the separated charges increases, which might be favourable for the ultimate conservation of energy. The more extensive energization of mature thylakoids was also indicated by a light-induced decrease in the thickness of the membranes upon illumination; a change which could not be detected in juvenile thylakoids.

Organization of Chlorophyll a in the Light-Harvesting Chlorophyll a/b Protein Complex as Shown by Circular Dichroism : Liquid Crystal-Like Domains.

The development of thylakoid stacking, accumulation of the light-harvesting chlorophyll a/b protein complex (LHCP), and the changes of circular dichroism (CD) which reflect the organization of chlorophyll molecules in greening thylakoids of bean Phaseolus vulgaris cv Red Kidney leaves were investigated.Chloroplasts formed under intermittent light contained large double sheets of membrane with extensive appression in addition to separate lamellae. Thylakoids of such chloroplasts were devoid of LHCP and exhibited a relatively small CD in the chlorophyll absorption region. Upon continuous illumination, the rearrangement of membranes to characteristic grana and the accumulation of the LHCP was accompanied by the gradual appearance of the very intense CD signal with peaks at 682 to 684 (+) and 665 to 672 nanometers (-). The magnitude of differential absorption was approximately 100 times larger than that of the chlorophyll a in solution. This suggests a superhelical liquid crystal-like organization for LHCP, a texture which can be altered by changes of the electric field in the photosynthetic membranes.

Orientation of emitting dipoles of chlorophyll A in thylakoids: considerations on the orientation factor in vivo.

Orientation angles of five emitting dipoles of chlorophyll a in thylakoids were estimated from low temperature fluorescence polarization ratio spectra of magnetically oriented chloroplasts. A simple expression is given also for the evaluation of data from linear dichroism measurements. It is shown that the Qy dipoles of chlorophylls lie more in the plane of the membranes and span a larger angular interval than was previously thought. Values for the orientation factor are calculated using various models corresponding to different degrees of local order of the Qy dipoles of chlorophylls in the thylakoid. We show that the characteristic orientation pattern of the Qy dipoles of chlorophylls in the membrane, i.e., increasing dichroism toward longer wavelengths, may favour energy transfer between the antenna chlorophylls as well as funnel the excitation energy into the reaction centers.

Structural and functional stability of isolated intact chloroplasts.

The effect of in vitro ageing on the ultrastructure, electron transport, thermoluminescence and flash-induced 515 nm absorbance change of isolated intact (type A) chloroplasts compared with non-intact (types B and C) chloroplasts was studied.When stored in the dark for 18 h at 5°C, the structural characteristics of intact and non-intact chloroplasts were only slightly altered. The most conspicuous difference between the two was in the coupling of the electron transport which was tighter and more stable in intact chloroplasts. Under dark-storage the activity of PS 2* decreased and the -20°C peak of thermoluminescence increased at the expense of the emission at +25°C. These changes were less pronounced in the intact chloroplasts. PS 1 activity and the flash-induced 515 nm absorbance change were not affected by dark-storage.When kept in the light (80 W m(-2) (400-700 nm) for 1 h at 5°C), the thylakoid system of chloroplasts rapidly became disorganized. Although the initial activity of electron transport was much higher in intact chloroplasts, after a short period of light-storage the linear electron transport and the electron transport around PS 2 decreased in both types of preparations to the same low level. These changes were accompanied by an overall decrease of the intensity of thermoluminescence. PS 1 was not inhibited by light-storage, while the flash-induced 515 nm absorbance change was virtually abolished both in preparations of intact and non-intact chloroplasts.The data show that in stored chloroplast preparations intactness cannot be estimated reliably either by the FeCy test or by inspection under the electron microscope. These tests should be cross-checked on the level and coupling of the electron transport.

Functional characteristics of intact chloroplasts isolated from mesophyll protoplasts and bundle sheath cells of maize.

A procedure was developed to isolate mesophyll and bundle sheath chloroplasts of a high degree of intactness and low cross-contamination. Light-induced (14)CO2 fixation of isolated chloroplasts was similar to that of protoplasts and cells in that it was low and was stimulated by the addition of exogenous substrates. O2 evolution was absent in both bundle sheath chloroplasts and cells. The flash-induced 515 nm absorbance change of intact mesophyll chloroplasts showed a biphasic rise, previously known to be a characteristic only of intact algae. With bundle sheath chloroplasts or cells, no 515 nm signal could be detected. In the presence of 10 µmol l(-1) phenazine methosulphate, bundle sheath chloroplasts exhibited a flash-induced 515 nm signal with a monophasic rise and amplitude comparable to that of the mesophyll chloroplasts. A similar signal was obtained with bundle sheath chloroplasts suspended in an extract prepared from the mesophyll tissue. Both the substrate stimulation of the CO2 fixation and the reconstitution of the 515 nm signal in bundle sheath chloroplasts by the mesophyll extract indicate the requirement of cooperation between the mesophyll and bundle sheath cells of maize leaves.