Extraction of chlorophyll a from Tetradesmus obliquus-a method upgrade.

Nowadays, the use of algae is prevalent for both industrial and agricultural purposes. The determination of chlorophyll (Chl) content is a commonly used method for estimating the phytoplankton abundance in different water bodies or biomass density of algal cultures. The aim of the present work is to optimise the efficiency of the Chl extraction from the green alga Tetradesmus obliquus using methanol as extracting solvent. The extraction efficiency was estimated by measuring the Chl a concentration of the extracts using fluorescence spectroscopy. To increase the extraction yield, glass fibre filters with algal cells on top were treated with 10% (v/v) formalin prior to the extraction. We found that this pretreatment significantly enhanced the extraction yield of Chl without its chemical decomposition. We also found that the optimal cell concentration for Chl determination ranged from 1.44 × 104 to 3.60 × 105 cells/mL and the extraction efficiency was lower when the cell density of the culture was out of this range. These results highlight the importance of the optimization of the pigment extraction for the studied algal species.

Artificial tripartite symbiosis involving a green alga (Chlamydomonas), a bacterium (Azotobacter) and a fungus (Alternaria): morphological and physiological characterization.

A long-living artificial tripartite symbiosis involving a green alga (Chlamydomonas), a bacterium (Azotobacter) and a fungus (Alternaria) was established on carbon- and nitrogen-free medium. The basis of the interdependence is the complementation of photosynthetic CO2 assimilation and atmospheric nitrogen fixation. Green color of the colonies indicated that the algal cells had enough nitrogen to synthesize chlorophylls. The chlorophyll content was nearly 40% of the control cells. The relatively high rate of photosynthetic oxygen evolution proved that nitrogen was effectively used for building up a well functioning photosynthetic apparatus. This was supported by the analysis of photosystems and ultrastructural investigations. In comparison with degreened algae cultured on nitrogen-free medium, the chloroplasts in the symbiont algal cells contained a well-developed, stacked thylakoid membrane system without extreme starch or lipid accumulation. The occurrence of the fungus in the association greatly increased the chlorophyll content. Far fewer types of amino acids were excreted by the tripartite cultures than by pure cultures. Cystathionine, which is a common intermediate in the sulfur-containing amino acid metabolism, was produced in high quantities by the tripartite symbiosis. This can mostly be attributed to the activity of the fungus.

Disintegration of the prolamellar body structure at high concentrations of Hg2+.

The effects of high concentrations of Hg (2+) (10 (-2) M and 10 (-3) M) were investigated on the ultrastructure and on the light-induced transformation of isolated prolamellar bodies (PLBs) of dark-grown wheat leaves. Our earlier work on wheat leaf homogenates ( , Plant Biology 6, 358 - 368) showed that, depending on the concentration, Hg (2+) reacts with protochlorophyllide, NADPH and the NADPH : protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) enzyme and induces disaggregation of the macrodomain structure of this latter. Spectroscopic analyses confirmed that 15 min incubation with 10 (-2) M Hg (2+) at 4 degrees Celsius completely inhibited the activity of POR also in isolated PLBs. Ultrastructural investigations revealed the loosening of the PLB structure in the Hg (2+)-treated sample, i.e., intensive vesicle formation on the surface of the PLB membranes. The hexagonal geometry of the inner lattice was not disturbed, however, the unit cell size significantly increased. The disruption of the PLB membranes upon irradiation was studied after 40 min incubation with 10 (-3) M Hg (2+) at 4 degrees Celsius and a subsequent irradiation for 40 min at 20 degrees Celsius. Equimolar concentrations (10 (-3) M) of NADPH and Hg (2+) were added to the samples 10 min prior or after the addition of Hg (2+). Our results suggest that Hg (2+) accelerates the disruption of the PLB membranes and that NADPH can only partially prevent this process. These membrane transformations were similar to those observed in the initial steps of the Shibata shift of control samples.

Hg(2+) reacts with different components of the NADPH : protochlorophyllide oxidoreductase macrodomains.

The molecular background of Hg (2+)-induced inhibition of protochlorophyllide (Pchlide) photoreduction was investigated in homogenates of dark-grown wheat leaves. Our earlier work showed that 15 min incubation with 10 (-2) M Hg (2+) completely inhibits the activity of NADPH : Pchlide oxidoreductase ( ). Detailed analysis of spectra recorded at 10 K indicated the appearance of emission bands at 638 and 650 nm, which are characteristic for NADP (+)-Pchlide complexes. Fluorescence emission spectra recorded with different excitation wavelengths, fluorescence lifetime measurements and the analysis of acetone extractions revealed that Hg (2+) can also react directly with Pchlide, resulting in protopheophorbide formation. At 10 (-3) M Hg (2+), the phototransformation was complete but the blue shift of the chlorophyllide emission band speeded up remarkably. This indicates oxidation of the NADPH molecules that have a structural role in keeping together the etioplast inner membrane components. We suggest a complex model for the Hg (2+) effect: depending on concentration it can react with any components of the NADPH : Pchlide oxidoreductase macrodomains.

Tissue specific protochlorophyll(ide) forms in dark-forced shoots of grapevine (Vitis viniferaL.).

Cuttings of grapevine (Vitis vinifera L. cv. Chardonnay) were dark-forced at least three weeks. Pigment contents, 77 K fluorescence emission, excitation spectra of the leaves, petioles, stems, transmission electron micrographs of the etioplasts from leaves, the chlorenchyma tissues of the stems were analysed. The dark-grown leaves, stems contained 8 to 10, 3 to 5 mug/g fresh weight protochlorophyllide, its esters, respectively. HPLC analysis showed that the molar ratio of the unesterified, esterified pigments was 7:3 in the shoot developed in darkness. The dark-forced leaves contained carotenoids identified as: neoxanthin, violaxanthin, antheraxanthin, lutein, beta-carotene. Detailed analyses of the fluorescence spectra proved that all tissues of the dark-forced shoots had protochlorophyllide or protochlorophyll forms with emission maxima at 628, 636, 644, 655, 669 nm. The 628, 636 nm emitting forms were present in all parts of the dark-forced shoot, but dominated in the stems, which may indicate an organ specificity of the etioplast development. Variations in the distribution of the pigment forms were even found in the different tissues of the stem. The subepidermal layers were more abundant in the 655 nm form than the parenchyma cells of the inner part of the cortex, the pith. In the latter cells, the plastid differentiation stopped in intermediary stages between proplastids, etioplasts. The plastids in the subepidermal layers had developed prolamellar body structures, which were similar to those of etiolated leaves. The results highlight the importance of organ-, tissue specificity of plastid differentiation for chlorophyll biosynthesis, greening of different plant organs.

Spectroscopic analysis of desiccation-induced alterations of the chlorophyllide transformation pathway in etiolated barley leaves.

Effects of water deficit on the chlorophyllide (Chlide) transformation pathway were studied in etiolated barley (Hordeum vulgare) leaves by analyzing absorption spectra and 77-K fluorescence spectra deconvoluted in components. Chlide transformations were examined in dehydrated leaves exposed to a 35-ms saturating flash triggering protochlorophyllide (Pchlide) and Chlide transformation processes. During the 90 min following the flash, we found that dehydration induced modifications of Chlide transformations, but no effect on Pchlide phototransformation into Chlide was observed. During this time, content of NADPH-Pchlide oxydoreductase in leaves did not change. Chlide transformation process in dehydrated leaves was characterized by the alteration of the Shibata shift process, by the appearance of a new Chlide species emitting at 692 nm, and by the favored formation of Chl(ide) A(668)F(676). The formation of Chl(ide) A(668)F(676), so-called "free Chlide," was probably induced by disaggregation of highly aggregated Chlide complexes. Here, we offer evidence for the alteration of photoactive Pchlide regeneration process, which may be caused by the desiccation-induced inhibition of Pchlide synthesis.

Light- and cold-stress effects on the greening process in epicotyls and young stems of red oak (Quercus rubra) seedlings.

Protochlorophyllide (Pchlide) and protochlorophyll (Pchl) were found in epicotyls of 14-day-old dark-germinated seedlings and in 100-day-old dark-grown stems of red oak (Quercus rubra L.). Fluorescence spectroscopy measurements of epicotyls at 77 K showed that the majority of Pchlide and Pchl is present as a shorter wavelength-emitting monomer with a fluorescence emission maximum at 629-631 nm. A small amount of a monomeric form emitting at 635-636 nm was also present. Minor amounts of Pchlide were aggregated into larger complexes with fluorescence emission maxima at 640, 644-646 and 652-654 nm, as seen in etiolated leaves. Flash illumination transformed the 652-654-nm-emitting form to chlorophyllide, but not those forms with emission maxima at 629-631, 635-636 and 644-646 nm. These shorter wavelength-emitting forms were transformed to chlorophyllide by continuous illumination, but the process took several hours. Epicotyls and young stems were light sensitive, with exposure to full daylight causing strong pigment bleaching and tissue destruction. Complete greening took place only at low irradiances. Light sensitivity was greater at 4 degrees C than at room temperature. We conclude that the monomeric arrangement of the pigments accounted for the light and temperature sensitivity of the greening process in epicotyls and stems.

Protochlorophyllide and chlorophyll forms in dark-grown stems and stem-related organs.

Protochlorophyllide contents and the spectral properties together with photoactivities of native protochlorophyllide forms have been studied in dark-forced stems of 26 and epicotyls or hypocotyls of 9 plant species. The 77 K fluorescence emission spectra show that a form emitting at 629-631 nm is general in these organs. Besides this short-wavelength form, other protochlorophyllide forms emitting at 636, 645 and around 650-655 nm are found with various relative amplitudes. The pigment contents show good correlation with the ratio of short- to long-wavelength forms, i.e., the higher this ratio is, the less protochlorophyllide is detected. In addition to protochlorophyllide, several dark-grown plants also contain chlorophylls. In some cases only one chlorophyll form appears with emission maximum at 678-680 nm; other plants have forms characteristic of the fully developed photosynthetic apparatus (with maxima at 685, 695 and 730-740 nm). Flash illumination can transform only the 645 and 650-655 nm protochlorophyllide forms, the shorter-wavelength-emitting forms being inactive. Plant species with dominating 629-636 nm protochlorophyllide forms cannot accumulate chlorophyll on continuous illumination of natural intensity, and they became photodamaged. The structural or molecular background of the appearance of the different protochlorophyllide and chlorophyll forms and the reasons for their photosensitivity are discussed.

The two spectroscopically different short wavelength protochlorophyllide forms in pea epicotyls are both monomeric.

The spectral properties of the protochlorophyllide forms in the epicotyls of dark-grown pea seedlings have been studied in a temperature range, from 10 to 293 K with conventional fluorescence emission and excitation spectroscopy as well as by fluorescence line narrowing (FLN) at cryogenic temperatures. The conventional fluorescence techniques at lower temperatures revealed separate bands at 628, 634-636, 644 and 655 nm. At room temperature (293 K) the 628 and 634-636 nm emission bands strongly overlapped and the band shape was almost independent of the excitation wavelength. Under FLN conditions, vibronically resolved fluorescence spectra could be measured for the 628 and 634-636 nm bands. The high resolution of this technique excluded the excitonic nature of respective excited states and made it possible to determine the pure electronic (0,0) range of the spectra of the two components. Thus it was concluded that the 628 and 634-636 nm (0,0) emission bands originate from two monomeric forms of protochlorophyllide and the spectral difference is interpreted as a consequence of environmental effects of the surrounding matrix. On the basis of earlier results and the data presented here, a model is discussed in which the 636 nm form is considered as an enzyme-bound protochlorophyllide and the 628 nm form as a protochlorophyllide pool from which the substrate is replaced when the epicotyl is illuminated with continuous light.

Selective inhibition of chlorophyll biosynthesis by nicotinamide.

Rhodobacter sphaeroides grown in the presence of nicotinamide excreted bacteriochlorophyll precursors, 2,4-divinyl protochlorophyllide (DV-Pchlide) and a small amount of 2-monovinyl protochlorophyllide (MV-Pchlide). Accumulation of these pigments indicates that nicotinamide inhibited the bacteriochlorophyll biosynthetic pathway site-specifically between DV-Pchlide and MV-Pchlide. This phenomenon is also observed in an aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114. Among 12 nicotinamide derivatives and isomers tested, only nicotinamide was effective, indicating that in addition to the completeness of the pyridine ring skeleton at positions 1 to 3, the carboxylic acid amide group is essential for this inhibition. The technique described in this report permits the simple preparation of large quantities of DV-Pchlide.

Fluorescence spectroscopy of iron-deficient plants.

Iron deficient cucumber (Cucumis sativus L.) plants and such differently supplied with iron were studied by low-temperature fluorescence emission and excitation spectroscopy. Iron deficiency caused a relative decrease and a blue-shift of the 733 nm emission and a decrease or disappearance of the 700 and 707-710 nm excitation bands which are considered to belong to chlorophyll forms of PSI. The process of iron uptake was observed after readdition of iron to previously iron-stressed plants. Iron-deficient plants (with green cotyledon) were treated with the chelator EDTA which treatment resulted in a small increase of the chlorophyll content but the fluorescence spectra of these plants differed from those of plants supplied with iron: they had blue-shifted maxima. The data presented show that fluorescence spectroscopy is a very sensitive and suitable method for studying iron deficiency, observing - indirectly - iron uptake and its utilization in plants.

The role of a long-wavelength pigment form in the chlorophyll biosynthesis.

Photoconversion of protochlorophyllide650 form was observed in etiolated leaves illuminated with long-wavelength-690 nm-light. This process showed Shibata shift and was found to have a strong temperature dependence between 20 and -40°C. The low rate of reaction, the strong temperature dependence and calculations on the spectral overlap integral of absorption and fluorescence bands in this spectral region indicate that the phototransformation of the 650 nm form of protochlorophyllide may be caused by a back energy migration from a long-wavelength pigment form absorbing around 690 nm; this pigment form is probably a long-wavelength form of protochlorophyll/ide.

Protochlorophyll forms with different molecular arrangements.

Spectral properties of protochlorophyll ()PChl) forms were investigated in solid-film model systems by absorption fluorescence and circular dichroism (CD) spectroscopy. The solid films were prepared from diethyl ether solution of PChl on a cover glass surface by evaporation of the solvent. After preparation the films usually showed an absoprtion maximum at 635 nm or in some cases at 640 nm. The PChl form with 635 nm absorption maximum had no CD signal, whilst the films with absorption maximum at 640 nm gave an intense negative CD band at about 640 nm and a positive one at 668 nm. The treatment of the films with ammonia or acetone vapour resulted in a red shift of the absorption maximum from 635 nm or 640 nm to 650 nm. The study of the CD spectra of the films with different PChl forms showed that, depending on the treatment, forms of PChl with similar absorption and fluorescence spectra, but with opposite CD signals, can exist. It is suggested that the differences of the CD spectra are mainly due to different arrangements of the aggregates.