We present the first experimental results on salinity tolerance and regulation mechanisms in the genus Tolypella. The two species investigated, T. nidifica and T. glomerata, regulate turgor pressure with almost complete effectiveness by adjustment of K + and Cl - concentrations. Sucrose is also involved. The mechanism is basically identical to the mechanism of turgor pressure regulation previously identified in representatives of the genera Chara and Lamprothamnium. Since Chara and Lamprothamnium on the one hand and Tolypella on the other belong to different phylogenetic branches that separated early in the geological history of the Characeae, the K + regulation mechanism can be assumed to represent an ancient pattern derived from a salt-tolerant common ancestor. Furthermore, our experiments provide evidence that salinity is a limiting factor for fertility in both T. nidifica and T. glomerata. Although the onset of gametangia covers the whole range of salinities tested here (0-29 psu), 12 psu was the inhibitory level for the formation of mature oospores. Fertilization is probably disturbed by an increase in salinity. An inability to reproduce sexually under euryhaline conditions could explain why the distribution of the two species is restricted to oligo- and mesohaline environments, despite the wide range of salinity tolerance of their vegetative apparatus.

Effects of salinity on turgor pressure and fertility in Tolypella (Characeae)

We applied the endocytic markers FM1-43, FM4-64 and filipin to internodal cells of the green alga Chara corallina. Both FM dyes stained stable, long-living plasma membrane patches with a diameter of up to 1 microm. After 5 min, FM dyes labeled cortical, trembling structures up to 500 nm in size. After 15 min, FM dyes localized to endoplasmic organelles up to 1 microm in diameter, which migrated actively along actin bundles or participated in cytoplasmic mass streaming. After 30-60 min, FM fluorescence appeared in the membrane of small, endoplasmic vacuoles but not in that of the central vacuole. Some of the FM-labeled organelles were also stained by neutral red and lysotracker yellow, indicative of acidic compartments. Filipin, a sterol-specific marker, likewise labeled plasma membrane domains which co-localized with the FM patches. However, internalization of filipin could not be observed. KCN, cytochalasin D, latrunculin B and oryzalin had no effect on size, shape and distribution of FM- and filipin-labeled plasma membrane domains. Internalization of FM dyes was inhibited by KCN but not by drugs which interfere with the actin or microtubule cytoskeleton. Our data indicate that the plasma membrane of characean internodal cells contains discrete domains which are enriched in sterols and probably correspond to clusters of lipid rafts. The inhibitor experiments suggest that FM uptake is active but independent of actin filaments, actin polymerization and microtubules. The possible function of the sterol-rich, FM labeled plasma membrane areas and the significance of actin-independent FM internalization (via endocytosis or energy-dependent flippases) are discussed.

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FM dyes label sterol-rich plasma membrane domains and are internalized independently of the cytoskeleton in characean internodal cells.

We investigated the identity and distribution of cortical domains, stained by the endocytic marker FM 1-43, in branchlet internodal cells of the characean green algae Chara corallina and Chara braunii. Co-labeling with NBD C(6)-sphingomyelin, a plasma membrane dye, which is not internalized, confirmed their location in the plasma membrane, and co-labelling with the fluorescent pH indicator Lysotracker red indicated an acidic environment. The plasma membrane domains co-localized with the distribution of an antibody against a proton-translocating ATPase, and electron microscopic data confirmed their identity with elaborate plasma membrane invaginations known as charasomes. The average size and the distribution pattern of charasomes correlated with the pH banding pattern of the cell. Charasomes were larger and more frequent at the acidic regions than at the alkaline bands, indicating that they are involved in outward-directed proton transport. Inhibition of photosynthesis by DCMU prevented charasome formation, and incubation in pH buffers resulted in smaller, homogenously distributed charasomes irrespective of whether the pH was clamped at 5.5 or 8.5. These data indicate that the differential size and distribution of charasomes is not due to differences in external pH but reflects active, photosynthesis-dependent pH banding. The fact that pH banding recovered within several minutes in unbuffered medium, however, confirms that pH banding is also possible in cells with evenly distributed charasomes or without charasomes. Cortical mitochondria were also larger and more abundant at the acid bands, and their intimate association with charasomes and chloroplasts suggests an involvement in carbon uptake and photorespiration.

Plasma Membrane Domains Participate in pH Banding of Chara Internodal Cells

In this chapter, we explore the origins and evolutionary history of sucrose synthesis in plants. Our journey begins with the photosynthetic proteobacteria and cyanobacteria, which were probably the first organisms to synthesize this disaccharide sugar. Sucrose remained an obscure metabolite, used mainly for osmoregulation, until the endosymbiosis of a sucrose-synthesizing cyanobacterium gave rise to the chloroplasts of green plants. This unique event opened up new opportunities for sucrose, and ultimately led to its achieving global importance as one of the principal products of photosynthesis in plants. The rise of sucrose parallels the evolution of plants themselves, with the appearance of long distance vascular transport systems and the adoption of sucrose as a major transport sugar being key developments. During this process the two enzymes in the committed pathway of sucrose synthesis – sucrose-phosphate synthase (SPS) and sucrose-phosphatase (SPP) – evolved new structural and regulatory features, such as the phosphorylation sites involved in light-dark modulation and osmotic stress activation of SPS. We describe how these and other regulatory mechanisms act to coordinate the rate of sucrose synthesis in leaves with photosynthetic CO2 fixation, sucrose export and storage. As our evolutionary journey nears its end, we discover how gene duplications and divergence gave rise to the four SPS gene families – A, B, C and D – that we see in angiosperms today, and how plants have recruited each of the SPS isoforms for different purposes, although these may vary from species to species. Having charted the meteoric rise of sucrose, we finish with some speculation on why this sugar of humble origin came to achieve such prominence in the life of plants, concluding that the secrets of its success remain largely a mystery.

Photosynthetic Sucrose Biosynthesis: An Evolutionary Perspective

During the development of the antheridia of Chara species, dynamic changes in the occurrence and ultrastructure of plasmodesmata are observed which are closely correlated to particular developmental phases and presumably regulate the morphogenetic events in the antheridia. The disappearance of plasmodesmata between shield cells and between shied cells and the basal cell leads to a cessation in symplasmic transport around the antheridum and determines its concentric or centrifugal character via centrally situated capitular cells. Unplugged plasmodesmata are present between fully synchronously developing antheridial filament cells and obviously coordinate the development of the cells. In the middle phase of spermiogenesis, rough endoplasmic reticulum in antheridial filaments passes uncompressed through wide plasmodesmata and provides an additional transport pathway for developmental control factors. Plugged plasmodesmata link cells of different types or cells of the same type which are at different phases of cell cycle and guarantee their individual development. The plugging of plasmodesmata is a reversible process that depends on the morphogenetic situation. Plasmodesmata connecting the basal cell and the subbasal cell as well as the basal cell and capitular cells are transformed successively from the simple into the complex type and might be the pathways for an import of gibberellins and nutrients into the strong sink tissues of the developing antheridium. There is a symplasmic connection between the antheridum and the thallus via a basal cell. Prior to the initiation of spermatozoid differentiation (spermiogenesis), plasmodesmata connecting the basal cell with a subbasal cell and the basal cell with capitular cells are spontaneously broken, resulting in symplasmic isolation of the antheridium that is probably a signal which triggers the induction of spermatozoid differentiation. Premature plasmolytically evoked symplasmic isolation of the antheridium leads to the elimination of 1 to 2 cell cycles from the proliferative stage of spermatogenesis. Autoradiographic studies demonstrate that both natural and induced symplasmic isolation drastically decreases the entry of isotopically labeled gibberellic acid into antheridia of Chara species that may be the consequence of the elimination of the hormone's transport through plasmodesmata.

Plasmodesmal changes are related to different developmental stages of antheridia of Chara species.

In cuttings of Chara vulgaris consisting of 3–4 internodal cells with whorls (branchlets) transport of photoassimilates was investigated by 14C-labelling. The distribution of radioactivity in different component parts of the plant was shown as percentage and calculated in relation to fresh weight. Sucrose was identified as the main transported carbohydrate. In fertile Ch. vulgaris plants, developing oogonia and antheridia were important sinks for transported carbohydrates. Polarity of transport in the internodal cells (nutrients and assimilates) remains constant, as the main characteristic, from base to apex in fertile as well as in sterile plants. In sterile plants, the branchlets being the main source for photoassimilates and the apex the sink, transport from branchlets is mainly in the direction of the internodes. In the case of fertile plants gametangia are additional sinks redirecting transport of assimilates into the branchlets.

U. Winter

Papers

Source‐sink Characteristic of Photoassimilate Transport in Fertile and Sterile Plants of Chara vulgaris L.*