O. A. Gorelova

EFFECT OF NITROGEN STARVATION ON OPTICAL PROPERTIES, PIGMENTS, AND ARACHIDONIC ACID CONTENT OF THE UNICELLULAR GREEN ALGA PARIETOCHLORIS INCISA (TREBOUXIOPHYCEAE, CHLOROPHYTA) 1

Spectral properties of cell suspensions, individual cells, and extracts of the unicellular green alga Parietochloris incisa (Reisigl) Shin Watan. grown under low light were studied. Long‐term nitrogen (N) deprivation resulted in a decrease of chloroplast volume, appearance of numerous large cytoplasmic oil bodies, and the deposition of triacylglycerols with a high proportion of arachidonic acid. Chlorophylls a and b underwent a synchronous decline, whereas carotenoids (Car) showed a relative increase. Simultaneously, significant qualitative changes in the spectral properties of P. incisa individual cells, cell extracts, and cell suspensions were observed. To a large extent, the spectral changes observed in cell suspension could be attributed to a decrease in overall pigment content, leading to a gradual weakening of the so‐called package effect and accumulation of additional amounts of Car over chl, most probably, in oil bodies. Several optical characteristics of cell suspensions could serve as sensitive indicators of N‐deficiency in P. incisa. Furthermore, the absorption ratios, A476/A676 and A650/A676, showed close correlations with the Car‐to‐chl ratio and relative arachidonic acid (AA) content, respectively. The latter makes it possible to suggest that the increase in AA percentage in P. incisa proceeds in parallel with a decrease in cell chl content, accounting for the weakening of the package effect. N‐replenishment resulted in complete recovery of cell optical properties. The possible significance of the changes in cell ultrastructure, pigments, lipids, and optical properties is discussed with special reference to the ability of algae to adapt to and survive under conditions of long‐term nutrient deficiency.

Artificial Cyanobacterium-Plant Symbioses

Studies conducted during the last decade have significantly improved our knowledge of the differrent stages in formation and function of artificial associations involving cyanobacteria and furnished new data on the involvement of satellite bacteria in this process. The strenuous efforts to create artificial associations between important agricultural plants and nitrogen-fixing microorganisms have yielded promising results particularly for introducing heterocyst-forming cyanobacteria into the plant rhizosphere. Important progress has also been made in the induction of root paranodule and colonization of such para-nodules by microsymbionts.

Associations between the White Sea Invertebrates and Oxygen-Evolving Phototrophic Microorganisms

Eleven species of White Sea invertebrates (sponges, actinians, hydroids, polychaetes, and nudibranch mollusks) were tested for the presence of associated oxygen-evolving phototrophic microorganisms (OPM) (microalgae and cyanobacteria). Two main approaches were applied: (a) light and electron microscopy of intact samples and fixed preparations of invertebrates, and (b) isolation of microorganisms from samples of invertebrates after mild surface sterilization. The obtained results lead to conclusions on the formation of multicomponent associations by White Sea invertebrates and OPM based on the following data: (1) isolation of 27 cultures of OPM from eight species of invertebrates (sponges, hydroids, polychaete trochophore larva), (2) specificity of association between epibiontic communities of microorganisms and macroorganisms within the same biotope, and (3) spatial integration of micro- and macropartners resulting in the formation of morphological structures within the interorganismic contact zones.

Communication of cyanobacteria with plant partners during association formation

Data are presented on the physiological diagnostics of cyanobacterial communication with higher plants in natural symbioses (plant syncyanoses) and in model associations, as well as on the interaction of the partners without spatial integration. Emphasis is placed on changes in cyanobacterial features important for symbiogenesis. The multicomponent composition and the possible nature of the factors that enable partner communications are discussed with hormogonia formation and taxis as an example.

Similarity and diversity of the Desmodesmus spp. microalgae isolated from associations with White Sea invertebrates.

Similarity and diversity of the phenotype and nucleotide sequences of certain genome loci among the single-celled microalgae isolated from White Sea benthic invertebrates were studied to extend the knowledge of oxygenic photoautotrophs forming microbial communities associated with animals. We compared four Desmodesmus isolates (1Hp86E-2, 1Pm66B, 3Dp86E-1, 2Cl66E) from the sponge Halichondria panicea, trochophore larvae of the polychaete Phyllodoce maculata, and the hydroids Dynamena pumila and Coryne lovenii, respectively. The microalgae appeared to be very similar featuring the phenotypic and genetic traits characteristics of unicellular representatives of the genus Desmodesmus. At the same time, isolates from different animal species displayed certain differences in (i) the epistructure morphology; (ii) type and number of the inclusions such as interthylakoid starch grains and cytoplasmic oil bodies and (iii) fatty acid composition; in Desmodesmus sp. 1Hp86E-2, these differences were most pronounced. Phylogenetic analysis based on ITS1-5.8S rRNA-ITS2 and rbcL sequences showed that all isolates studied differ from known classified representatives of Desmodesmus combining a deletion in the conservative 5.8S rRNA gene and long AC-microsatellite repeats in the ITS1 whereas 1Hp86E-2 represented a distinct branch within this group.

Green microalgae isolated from associations with white sea invertebrates

Endophotosymbionts (cyanobacteria, microalgae, or their functionally active chloroplasts) were found in mollusks, sponges, corals, anemones, freshwater hydra, worms, and ascidians [1–2]. Associations of colonial hydroids with epibiotic microalgae and cyanobacteria were previously described [3 ⎯5]. It is known that isolation of animal microsymbionts is sel� dom successful. The information on the isolation of phototrophic microrganisms associated with inverte� brates of the high latitude seas, especially the White Sea, is limited and mainly refers to cyanobacteria [4]. The goal of the present work was to study the microalgae isolated from associations with the White Sea invertebrates and to characterize their morphol� ogy, ultrastructure, and the composition of pigments and fatty acids. Microalgae analyzed in the study were isolated from the benthic animals collected in the region of the Moscow State University White Sea Biological Station ( 66°34′N, 33°08′E) in the Kandalaksha Bay of the White Sea. Microalgae were isolated from the inverte� brates with green zones containing red autolumines� cent bodies within their tissues or covers. Isolation and microscopy were carried out as described [4, 5]. Fatty acid and pigment analysis was carried out according to the method in [6]; molecular genetic analysis was car� ried out as described in [7].

Ultrastructure of cyanobacterium Nostoc sp. f. Blasia cell forms in persisting populations

Cell clusters formed in persistent populations of Nostoc sp. f. Blasia, a cyanobacterium capable of cell differentiation, under prolonged storage in the dark at low temperatures were studied for the first time. Cell reorganization was observed, including changes in the ultrastructure of thylakoids, the cell wall peptidoglycan layer, and carboxysomes. Subcellular structures involved in intercellular communication within the clusters were revealed (structures similar to microplasmodesms and contact pores, secretory vesicles, etc.) Persistence of cyanobacterial populations was concluded to result from formation not only of specialized dormant cells (akinetes), but also L-forms, as well as from the modification changes of the clustered vegetative cells. A cluster containing the vegetative cells and L-like forms within a common intercellular matrix is considered a structural unit at the supracellular level, which is responsible for survival of cyanobacterial populations when mass akinete formation does not occur.

Formation of giant and ultramicroscopic forms of Nostoc muscorum CALU 304 during cocultivation with Rauwolfia tissues

The study of heteromorphic Nostoc muscorum CALU 304 cells, whose formation was induced by 6- to 7-week cocultivation with the Rauwolfia callus tissues under unfavorable conditions, revealed the occurrence of giant cell forms (GCFs) with a volume which was 35–210 times greater than that of standard cyanobacterial cells. Some GCFs had an impaired structure of the murein layer of the cell wall, which resulted in a degree of impairment of the cell wall ranging from the mere loss of its rigidity to its profound degeneration with the retention of only small peptidoglycan fragments. An analysis of thin sections showed that all GCFs had enlarged nucleoids. The photosynthetic membranes of spheroplast-like GCFs formed vesicles with contents analogous to that of nucleoids (DNA strands and ribosomes). About 60% of the vesicles had a size exceeding 300 nm. With the degradation of GCFs, the vesicles appeared in the intercellular slimy matrix. It is suggested that the vesicles are analogous to elementary bodies, which are the minimal and likely primary reproductive elements of L-forms. The data obtained in this study indicate that such L-forms may be produced in the populations of the cyanobionts of natural and model syncyanoses. Along with the other known cyanobacterial forms induced by macrosymbionts, L-forms may represent specific adaptive cell forms generated in response to the action of plant symbionts.

Surface Ultrastructure of the Heteromorphic Cells of Nostoc muscorumCALU 304 in a Mixed Culture with the RauwolfiaCallus Tissue

The ultrastructure of the heteromorphic cells (HMCs) of the cyanobacterium Nostoc muscorumCALU 304 grown in pure culture, monoculture, and a mixed culture with the Rauwolfiacallus tissue was studied. The comparative analysis of the cell surface of HMCs, the frequency of the generation of cell forms with defective cell walls (DCWFs), including protoplasts and spheroplasts, and the peculiarities of their ultrastructure under different growth conditions showed that, in the early terms of mixed incubation, the callus tissue acts to preserve the existing cyanobacterial DCWFs, but begins to promote their formation in the later incubation terms. DCWFs exhibited an integrity of their protoplasm and were metabolically active. It is suggested that structural alterations in the rigid layer of the cell wall may be due to the activation of the murolytic enzymes of cyanobacteria and the profound rearrangement of their peptidoglycan metabolism caused by the Rauwolfiametabolites diffused through the medium. These metabolites may also interfere with the functioning of the universal cell division protein of bacteria, FtsZ. In general, the Rauwolfiacallus tissue promoted the unbalanced growth of the cyanobacterium N. muscorumCALU 304 and favored its viability in the mixed culture. The long-term mixed cultivation substantially augmented the probability of the formation of L-forms of N. muscorumCALU 304.

The Accumulation and Degradation Dynamics of Cyanophycin in Cyanobacterial Cells Grown in Symbiotic Associations with Plant Tissues and Cells

Five different artificial associations of cyanobacterial cells with the cells or tissues of nightshade and rauwolfia were studied. The associations grown on nitrogen-containing media produced heterocysts. Cyanobacterial cells in the associations retained their ability to take up combined nitrogen from the medium, to store it in the form of cyanophycin granules, and to use them in the process of symbiotic growth. The synthesis and degradation of cyanophycin granules in cyanobacterial cells were more active in the associations than in monocultures. In the symbiotic associations of Chlorogloeopsis fritschii ATCC 27193 with Solanum laciniatum cells and of Nostoc muscorum CALU 304 with the Rauwolfia serpentina callus, heterocysts were produced with a 3- to 30-fold higher cyanophycin content than in pure cyanobacterial cultures. In contrast, in the association of N. muscorum CALU 304 with the Solanum dulcamara callus, heterocysts were produced with a lower cyanophycin content than in the N. muscorum CALU 304 pure culture. The degradation of cyanophycin granules in N. muscorum CALU 304 cells grown in associations with plant tissues or cells was subjected to mathematical analysis. The activation of cyanophycin degradation and heterocyst differentiation in the associations N. muscorum CALU 304–R. serpentinaand C.fritschii–S. laciniatum was accompanied by an enhanced synthesis of the nitrogen-containing alkaloids in plant cells. The data obtained suggest that an integrated system of nitrogen homeostasis can be formed in symbiotic associations. Depending on the growth stage of an association, its plant member can either stimulate the accumulation of combined nitrogen in vegetative cyanobacterial cells in the form of cyanophycin granules, activate their degradation, or initiate the formation of heterocysts independently of the cyanobacterial combined nitrogen deprivation sensing-signaling pathway.