Simonetta Pancaldi

Frequently asked questions about in vivo chlorophyll fluorescence: practical issues

The aim of this educational review is to provide practical information on the hardware, methodology, and the hands on application of chlorophyll (Chl) a fluorescence technology. We present the paper in a question and answer format like frequently asked questions. Although nearly all information on the application of Chl a fluorescence can be found in the literature, it is not always easily accessible. This paper is primarily aimed at scientists who have some experience with the application of Chl a fluorescence but are still in the process of discovering what it all means and how it can be used. Topics discussed are (among other things) the kind of information that can be obtained using different fluorescence techniques, the interpretation of Chl a fluorescence signals, specific applications of these techniques, and practical advice on different subjects, such as on the length of dark adaptation before measurement of the Chl a fluorescence transient. The paper also provides the physiological background for some of the applied procedures. It also serves as a source of reference for experienced scientists.

Neochloris oleoabundans grown in enriched natural seawater for biodiesel feedstock: evaluation of its growth and biochemical composition.

The freshwater microalga Neochloris oleoabundans was used to study algal lipid production in enriched natural seawater, in order to assess its suitability as biodiesel feedstock. Optimal and nitrogen-stress (N-stress) conditions were analyzed. Under optimal conditions, the strains growth rate was 0.73 div day(-1) and the biomass concentration was 1.5 g L(-1), while it had a maximum lipid yield under N-stress conditions (lipid content: 26% of dry weigh and lipid productivity: 56 mg L(-1) day(-1)). Lipid accumulation was mainly due to a significant increase of triacylglycerol content. Neutral lipids were characterized by a dominance of monounsaturated fatty acids and displayed a fatty acid profile that is suitable for biodiesel. This work offers an interesting alternative for sustainable microalgal oil synthesis for biodiesel production without using freshwater resources. However, further studies are necessary in order to optimize the lipid productivities required for commercial biodiesel production.

Multiple transcription start sites of the carrot dihydrofolate reductase-thymidylate synthase gene, and sub-cellular localization of the bifunctional protein

The analysis of clones obtained by rapid amplification of the 5′ endand by primer extension of the mRNA for carrot bifunctionaldihydrofolate reductase-thymidylate synthase showed transcripts ofdiffering lengths that belonged to two sub-populations. The longertranscripts were found to contain a translation start site 147 ntupstream of, and in frame with, the one which is present in the shortertranscripts. The ORF that begins at this ATG codes for a protein of64714 Da, which is much larger than mature DHFR-TS subunit. TheN-terminusregion of this polypeptide shows features typical of plant transitpeptides. Immunogold labelling studies and immunorecognition of theplastid-containing sub-cellular fraction suggested a plastidiallocalisation of the bifunctional protein. Although plant cells wereshown to contain folate pools in plastids, in mitochondria and in thecytosol, few enzymes of the folate pathway have been associated with anysub-cellular compartment. Thus, this is the first indication for thepresence of an enzyme of the folate biosynthetic pathway in plastids.The longer transcripts revealed the presence of a TC microsatellite atthe 5′-untranslated end.

Effects of Congo red on wall synthesis and morphogenesis in Saccharomyces cerevisiae

Abstract Congo red, a stain which prevents polyglycan microfibril assembly, was assayed on growing cells of Saccharomyces cerevisiae. In the presence of 0.25 mg/ml of the chemical, yeast developed aberrant wall thickenings and malformed septa, and underwent several budding events without the occurrence of the separation between mother and daughter cells. It is suggested that Congo red may uncouple polymerization and crystallization of the wall constituents, primarily chitin, that normally form microfibrils. It is also propsed that the regular assembly of the chains into microfibrils is needed to give a correct rate and direction to the process of polyglycan synthesis.

Complementary UV-Absorption of Mycosporine-like Amino Acids and Scytonemin is Responsible for the UV-Insensitivity of Photosynthesis in Nostoc flagelliforme

Mycosporine-like amino acids (MAAs) and scytonemin are UV-screening compounds that have presumably appeared early in the history of life and are widespread in cyanobacteria. Natural colonies of the UV-insensitive Nostoc flagelliforme were found to be especially rich in MAAs (32.1 mg g DW−1), concentrated in the glycan sheath together with scytonemin. MAAs are present in the form of oligosaccharide-linked molecules. Photosystem II activity, measured using PAM fluorescence and oxygen evolution, was used as a most sensitive physiological parameter to analyse the effectiveness of UV-protection. Laboratory experiments were performed under controlled conditions with a simulated solar radiation specifically deprived of UV-wavebands with cut-off filters (295, 305, 320, 345 and 395 nm). The UV-insensitivity of N. flagelliforme was found to cover the whole UV-A (315–400 nm) and UV-B (280–320 nm) range and is almost certainly due to the complementary UV-absorption of MAAs and scytonemin. The experimental approach used is proposed to be suitable for the comparison of the UV-protection ability in organisms that differ in their complement of UV-sunscreen compounds. Furthermore, this study performed with a genuinely terrestrial organism points to the relevance of marine photoprotective compounds for life on Earth, especially for the colonization of terrestrial environments.

Morphophysiological analyses of Neochloris oleoabundans (Chlorophyta) grown mixotrophically in a carbon-rich waste product

Neochloris oleoabundans is considered one of the most promising oil-rich microalgae because of its ability to store lipids under nitrogen starvation. However, high biomass densities, required for applications on medium to large scale, are not reached in this condition of growth. As previous studies on other microalgae have shown that mixotrophy allows to obtain higher biomass in comparison to autotrophic cultures, we performed morphophysiological analyses in order to test the mixotrophic growth capability of N. oleoabundans. A carbon-rich manure derived from the apple vinegar production (AWP) was added to the medium. Cells were also cultivated under nutrient starvation (tap water), to observe the expected lipids accumulation, and combining AWP to water, to test the potential of this waste in a low-cost culture system. The results highlighted that AWP in the medium allowed to obtain the highest final cell density. Moreover, starch granules were stored inside chloroplast at the beginning of the experiment. The presence of AWP did not induce variations on light harvesting complex II (LHCII)–photosystem II (PSII) assembly, even if an interesting promotion of pigment synthesis in cells was observed. On the other hand, in starved cells, chloroplast degeneration, pigment content decrease, altered LHCII–PSII assembly and accumulation of high amount of lipid globules were observed, irrespective of the presence of AWP. The results suggest that mixotrophy promotes growth in N. oleoabundans and open up the possibility of using waste products from agri-food industries for this purpose. After growth, cells could be transferred under nutrient starvation to induce lipid accumulation.

Morphological anomalies induced by Congo red in Aspergillus niger

Aspergillus niger germ tubes were exposed for 6 h to 0.15 mg/ml of Congo red, a stain which prevents chitin microfibril assembly. The most evident alterations, detected under ultraviolet light and by transmission and scanning electron microscopy, concerned the hyphal tips which burst or, most frequently, expanded into bulges. In the latter structures, new hyphal tips appeared which, after giving rise to more or less developed hyphae, were themselves converted into new bulges. Therefore, segments derived from isotropic and polarized growth alternated in the organisms exposed to the dye.An interpretation of these abnormalities is advanced based upon the assumption that the maintainance of a regular gradient of wall viscosity in the hyphal extension zone depends primarily on the capability of glycan chains to form crystalline aggregates of increasing complexity.

Responses of Trapa natans L. floating laminae to high concentrations of manganese

Summary.The present study focuses on the responses of floating laminae of the Mn-tolerant hydrophyte Trapa natans L. to 1 mM Mn and their ability to accumulate the metal. Studies were carried out first on young floating laminae belonging to the second verticil of 30-day-old plants which originated from fruits that had been maintained in a 1 mM Mn-treated environment and again on the young floating laminae after 10 days of further treatment with 1 mM Mn. Mn storing was observed from the first days after germination, but only 10-day-treated laminae showed the capability to hyperaccumulate the element inside specialised cells (>20000 μg/g [dry weight]). Electron microscopy and the Folin–Ciocalteu reaction for phenolics revealed deposits of chelated material inside vacuoles of the first palisade layer and of idioblasts in the spongy tissue. X-ray microanalysis indicated that the deposits were Mn chelated with phenolic compounds. Numerous trichomes were observed at the lower epidermis of 10-day-treated laminae. They were rich in phenolics and characterised by Mn concretions at their base. As they are associated with a high concentration of the metal in culture water and sediments, trichomes may constitute a morphological differentiation for the secretion of Mn-chelating molecules into the culture water, as a probable “avoidance” mechanism. Finally, monitoring of the photosynthetic apparatus showed that photosynthetic function was not impaired, though differences in development occurred.

High salinity alters chloroplast morpho-physiology in a freshwater Kirchneriella species (Selenastraceae) from Ethiopian Lake Awasa

Plants differ in their ability to tolerate salt stress. In aquatic ecosystems, it is important to know the responses of microalgae to increased salinity levels, especially considering that global warming will increase salinity levels in some regions of the Earth, e.g., Ethiopia. A green microalga, Kirchneriella sp. (Selenastraceae, Chlorophyta), isolated from freshwater Lake Awasa in the Rift Valley, Ethiopia, was cultured in media amended with 0, 0.4, 1.9, 5.9, and 19.4 g NaCl·L(-1) adjusted with NaCl to five salinity levels adjusted with NaCl. Growth was monitored for 3 mo, then samples were collected for photosynthetic pigment determinations, microspectrofluorimetric analyses, and micro- and submicroscopic examinations. The best growth was found at 1.9 g NaCl·L(-1). In the chloroplast, excess NaCl affected the coupling of light harvesting complex II and photosystem II (LHCII-PSII), but changes in thylakoid architecture and in the PSII assembly state allowed sufficient integrity of the photosynthetic membrane. The mucilaginous capsule around the cell probably provided partial protection against NaCl excess. On the whole, the microalga is able to acclimate to a range of NaCl concentrations, and this plasticity indicates that Kirchneriella sp. may survive future changes in water quality.

Adaptive modifications of the photosynthetic apparatus in Euglena gracilis Klebs exposed to manganese excess

Summary.Asynchronous cultures of wild-type Euglena gracilis were tested for their morphophysiological response to 10 mM MnSO4. Growth was only moderately slowed (15%), while oxygen evolution was never compromised. Inductively coupled plasma analyses indicated that the Mn cell content doubled with respect to controls, but no signs of localised accumulation were detected with X-ray microanalysis. Evident morphological alterations were found at the plastid level with transmission electron microscopy and confocal laser scanning microscopy. An increase in the plastid mass, accompanied by frequent aberrations of chloroplast shape and of the organisation of the thylakoid system, was observed. These aspects paralleled a decrease in the molar ratio of chlorophyll a to b and an increase in the fluorescence emission ratio of light-harvesting complex II to photosystem II, the latter evaluated by in vivo single-cell microspectrofluorimetry. These changes were observed between 24 and 72 h of treatment. However, the alterations in the pigment pattern and photosystem II fluorescence were no longer observed after 96 h of Mn exposure, notwithstanding the maintenance of the large plastid mass. The response of the photosynthetic apparatus probably allows the alga to limit the photooxidative damage linked to the inappropriately large peripheral antennae of photosystem II. On the whole, the resistance of Euglena gracilis to Mn may be due to an exclusion–tolerance mechanism since most Mn is excluded from the cell, and the small amount entering the organism is tolerated by means of morphophysiological adaptation strategies, mainly acting at the plastid level.