Floridean Starch Grains Research Articles

Ultrastructural evolution, physiological traits, and carbon and nitrogen assimilation–related gene expression are compatible with the developmental transitions of parthenogenesis in Pyropia haitanensis (Bangiales, Rhodophyta)

Pyropia haitanensis has a typical parthenogenetic behavior under unisexual culture condition. In this study, ultrastructural evolution and physiological traits of photosynthesis and the expression characteristics of key enzyme genes related to carbon and nitrogen assimilation pathways at different developmental stages of parthenogenesis were analyzed. Ultrastructural observations showed that the gametophytic vegetative cells had cell wall, pyrenoids, abundant chloroplasts, and rarely scattered floridean starch grains. However, the appearance of gathered chloroplasts, plastoglobuli, large lipid droplets, pyrenoids encircled by numerous plastoglobuli, and an increased number of floridean starch grains in the cells during parthenogenesis provide neutral lipid and energy resources that are compatible with the developmental transitions. Physiologically, vegetative gametophytes possessed the highest Fv/Fm and Chl. a content and the lowest C/N ratio. However, compared with vegetative cell proliferation stage, the activities of RubisCO (ribulose bisphosphate carboxylase) and external CA (carbonic anhydrase) increased at early stages of parthenogenesis, and Fv/Fm and activities of PEPC (phosphoenolpyruvate carboxylase) and PEPCK (phosphoenolpyruvate carboxykinase) remained higher at later stages of parthenogenesis, suggesting that dynamic photosynthetic carbon fixation occurs in parthenogenesis. Quantitative real-time PCR analysis showed that selective regulation of key enzyme genes related to carbon and nitrogen assimilation pathways maintained stable carbon and nitrogen metabolism during developmental transitions of parthenogenesis. Moreover, during parthenogenesis, photosynthetic carbon fixation pathway potentially changed from C3 pathway at female gametophyte stage to C4 pathway at parthenosporophyte stage, which is similar to that of gametophytes and sporophytes in sexual reproduction. Meanwhile, the ammonia assimilation involved GDH pathway (glutamate dehydrogenase pathway) and GS/GOGAT cycle (glutamine synthetase/glutamate synthase cycle) in female gametophyte stage, while GS/GOGAT cycle was dominant in parthenosporophyte stage, suggesting that GS/GOGAT cycle may play an important role in the carbon and nitrogen balance during the developmental transitions of parthenogenesis. These results provide important information for revealing the adaptation strategy of parthenogenesis in Pyropia.

Combined effects of UVR and nutrients on cell ultrastructure, photosynthesis and biochemistry in Gracilariopsis longissima (Gracilariales, Rhodophyta)

The combined effects of ultraviolet radiation (UVR) and nutrients on the red macroalga Gracilariopsis longissima were analyzed for 14days in a 2×2 factorial experiment design. Cell ultrastructure, photosynthetic activity as in vivo chlorophyll a fluorescence and the accumulation of bioactive compounds as soluble proteins, starch, lipids, polyphenols and mycosporine-like amino acids (MAAs) were evaluated. Three main morphological alterations as defense mechanisms against UVR were observed, i.e., an increase in cell wall thickness, accumulation of floridean starch grains and multiplication of the number of layers of cortical cells. Interaction of light and nutrients affected photosynthetic variables, such as electron transport rate (estimator of photosynthetic capacity) and photoinhibition irradiance (Eopt), and also the level of N compounds (i.e., proteins and MAAs) and total carbon and polyphenol content. Other variables such as chlorophyll a, carotenoids, starch, lipids, antioxidant activity and cell viability were influenced by both factors, or only by light, but without interactive effects. G. longissima showed a high acclimation to elevated UVR and nutrients by the decrease of the photosynthetic rate and the increase in the accumulation of N compounds with photoprotection and antioxidant capacities. On the other hand, starch, lipid and polyphenols increased mainly in the presence of UVR under low nutrient availability. The biotechnological implications of the metabolic changes due to N availability and UVR in this species are discussed.

Influence of cadmium and salinity in the red alga Pterocladiella capillacea: cell morphology, photosynthetic performance and antioxidant systems

This study aimed to determine the effects of cadmium (Cd) on apical segments of Pterocladiella capillacea (S. G. Gmelin) Santelices & Hommersand. For 7 days, these segments were cultivated under different salinities (25, 35 and 45 psu) and different Cd concentrations (0.17–0.70 ppm Cd). The samples were processed for light and confocal microscopy to assess photosynthetic performance, CHN levels, protein levels, as well as NADH dehydrogenase, and catalase activities. Plants cultivated at 0.70 ppm showed a reduction in the number of floridean starch grains by cytochemical analysis. Chloroplast autofluorescence decreased with cadmium exposure, while the kinetic patterns of ETR-light curves showed no significant differences among the treatments. Treatments showed variations in CNH levels, but no clear trend was observed. The content of total soluble protein and mitochondrial NADH dehydrogenase activity varied significantly, depending on the cadmium concentration and salinity levels. However, catalase activity decreased only in samples exposed to both cadmium and salinity treatments. Based on these lines of evidence, the present results showed a higher sensitivity of P. capillacea to increasing levels of cadmium exposure. When intermediate concentrations of cadmium were associated with low salinity values, its toxic effect on the model alga was intensified. Abiotic factors interfered with the physiological ability of the alga to protect itself from metal toxicity, resulting in metabolic compromise and reduction of primary productivity, i.e., photosynthesis and carbohydrate accumulation in the form of starch granules.

Effects of eutrophic seawater and temperature on the physiology and morphology of Hypnea musciformis J. V. Lamouroux (Gigartinales, Rhodophyta).

As both food and source of a kappa-carrageenan, Hypnea musciformis represents a species of great economic interest. It also synthesizes substances with antiviral, anti-helminthic and anti-inflammatory potential and shows promise for use as a bioindicator of cadmium. In this study, we investigated the combined effects of seawater from three urbanized areas (area 1: natural runoff, NRA; area 2: urbanized runoff and sewage with treatment, RTA; area 3: urbanized runoff and untreated sewage, RUS) and three different temperatures (15, 25 and 30 °C) on the growth rate, photosynthetic efficiency, photosynthetic pigments and cell morphology of H. musciformis. After 4 days (96 h) of culture, the biomass of H. musciformis showed differences that fluctuated among the areas and temperature treatments. Specifically, the specimens cultivated in 35 °C had low values of ETRmax, α(ETR), β(ETR), and Fv/Fm photosynthetic parameters, as well as changes in cell morphology, with reduction in photosynthetic pigments and drastic reduction in growth rates. When combined with the extreme temperatures, high concentrations of ammonium ion in seawater effluent caused an inhibition of photosynthetic activity, as well as significant variation in chlorophyll a and carotenoid contents. As observed by light microscopy, the synergism between different temperatures and pollutants found in eutrophic waters caused changes in cellular morphology with increased cell wall thickening and decreased floridean starch grains. H. musciformis also showed important changes in physiological response to each factor independently, as well as changes resulting from the synergistic interaction of these factors combined. Therefore, we can conclude that extreme temperature combined with the effect of eutrophic waters, especially RUS, caused distinct morphological and physiological changes in the red alga H. musciformis.

Effects of ultraviolet radiation (UVA+UVB) on young gametophytes of Gelidium floridanum: growth rate, photosynthetic pigments, carotenoids, photosynthetic performance, and ultrastructure.

This study investigated the effects of radiation (PAR+UVA+UVB) on the development and growth rates (GRs) of young gametophytes of Gelidium floridanum. In addition, photosynthetic pigments were quantified, carotenoids identified, and photosynthetic performance assessed. Over a period of 3 days, young gametophytes were cultivated under laboratory conditions and exposed to photosynthetically active radiation (PAR) at 80 μmol photons m(-2) s(-1) and PAR+UVA (0.70 W m(-2))+UVB (0.35 W m(-2)) for 3 h per day. The samples were processed for light and electron microscopy to analyze the ultrastructure features, as well as carry out metabolic studies of GRs, quantify the content of photosynthetic pigments, identify carotenoids and assess photosynthetic performance. PAR+UVA+UVB promoted increase in cell wall thickness, accumulation of floridean starch grains in the cytoplasm and disruption of chloroplast internal organization. Algae exposed to PAR+UVA+UVB also showed a reduction in GR of 97%. Photosynthetic pigments, in particular, phycoerythrin and allophycocyanin contents, decreased significantly from UV radiation exposure. This result agrees with the decrease in photosynthetic performance observed after exposure to ultraviolet radiation, as measured by a decrease in the electron transport rate (ETR), where values of ETRmax declined approximately 44.71%. It can be concluded that radiation is a factor that affects the young gametophytes of G. floridanum at this stage of development.

Ultrastructural, morphological, and molecular characterization of Colaconema infestans (Colaconematales, Rhodophyta) and its host Kappaphycus alvarezii (Gigartinales, Rhodophyta) cultivated in the Brazilian tropical region

Endophytic and epiphytic infections have caused serious problems for Kappaphycus farmers, such as reduction in biomass production and decrease in the yield and quality of carrageenan. During environmental monitoring from January 2011 to December 2012, along Pitimbu Beach, Paraiba State, northeastern Brazil, drifting thalli of Kappaphycus alvarezii (Gigartinales, Rhodophyta) were detected with red spots, apparently caused by epiphytic/endophytic infections. Therefore, drifting thalli of K. alvarezii farmed along the northeastern Brazilian coast were cultured in the laboratory and submitted to molecular and morphological analyses to identify and characterize the causative agent and its effects on the cellular structure and ultrastructure of the host alga K. alvarezii that was found to be infected by the endophyte Colaconema infestans (Colaconematales) identified through morphological and rbcL molecular evidence. Infected thalli of K. alvarezii were processed and analyzed through light, transmission electron, and scanning electron microscopy. Alterations were observed in morphology and cellular organization, including structural changes of chloroplasts and decrease in floridean starch grains, along with increased cell wall thickness. Therefore, while no outbreak has been reported, the discovery of C. infestans infection in drifting thallus of K. alvarezii suggests a potential threat to its cultivation that should be monitored.

Metabolic and cellular alterations induced by diesel oil in Hypnea musciformis (Wulfen) J. V. Lamour. (Gigartinales, Rhodophyta)

Understanding the mechanisms by which marine organisms cope to environmental stressful conditions is a fundamental question for ecotoxicology. We examined biochemical and cellular responses of Hypnea musciformis exposed in vitro to four concentrations of diesel oil (0 (control), 0.001, 0.01, 0.1, and 1 % v/v) for 0, 30 min, 1, 12, and 24 h. Chl a content decreased after exposure for 1, 12, and 24 h. Carotenoids increased after 30 min and 12 h and decreased after 1 and 24 h. Phenolic content was lower in samples exposed to 0.1 and 1 % of diesel oil. After treatment, samples showed a decrease in floridean starch grains, cell volume and autofluorescence intensity, changes in chloroplast morphology, and on the surface topography of the cell wall. These findings provide preliminary but important results on biochemical and cellular responses of H. musciformis when exposed to diesel oil and could be considered as tools for monitoring oil spills.

Changes in ultrastructure and cytochemistry of the agarophyte Gracilaria domingensis (Rhodophyta, Gracilariales) treated with cadmium

The agarophyte macroalgae Gracilaria domingensis (Kützing) Sonder ex Dickie is widely distributed along the Brazilian coast. While this species produces agarana, it is more important in the human diet. Therefore, the present study aimed to evaluate the biological effects of cadmium on its morphology and cellular organization. To accomplish this, the effects of cadmium in apical segments of G. domingensis were examined in vitro. Over a period of 16 days, the segments were cultivated and exposed to photosynthetically active radiation (PAR) at 80 μmol photons m(-2) s(-1), with cadmium treatments in doses of 100, 200 and 300 μM. The samples were processed for light, transmission and scanning electron microscopy. Histochemical analyses included Toluidine Blue for acidic polysaccharides, Coomassie Brilliant Blue for total protein, and Periodic Acidic Schiff for neutral polysaccharides. In all cadmium treatments, cytochemical analysis showed 1) metachromatic granulation in vacuole and lenticular thickness of the cell wall, 2) a higher concentration of cytoplasmic organelles, and 3) an increase in the number of floridean starch grains. Cadmium also caused changes in the ultrastructure of cortical and subcortical cells, including increased cell wall thickness and vacuole volume, as well as the destruction of chloroplast internal organization and increased number of plastoglobuli. In addition, treated plants showed a gradual increase in surface roughness, apparently the result of cadmium absorption. Taken together, these findings strongly suggested that cadmium negatively affects the agarophyte G. domingensis, posing a threat to the vitality of this plant species as a supplement in the human diet.

Responses of the macroalgae Hypnea musciformis after in vitro exposure to UV-B

The in vitro effects of UVBR were investigated in apical segments of Hypnea musciformis. The plants were cultivated and exposed to photosynthetically active radiation (PAR) at 80μmolphotonsm−2s−1 and PAR+UVBR at 1.6Wm−2 at 3h per day for 7 days. Toluidine Blue reaction showed metachromatic granulations in vacuole, and Periodic Acid Schiff stain showed a decrease in the number of floridean starch grains. UVBR also caused changes in the ultrastructure of cortical cells, which included increased thickness of the cell wall, reduced intracellular spaces, changes in the cell contour, destruction of chloroplast internal organization, and rough endoplasmic reticula increase. The algae cultivated under PAR-only showed growth rates of 9.7%day−1, while algae exposed to PAR+UVBR grew only 3.2%day−1. Furthermore, compared with control algae, phycobiliprotein contents (phycoerythrin, phycocyanin, and allophycocyanin) were observed to decrease after PAR+UVBR. However, chlorophyll a levels were not significantly different (ANOVA, P=0.52) after exposure to PAR+UVBR. As a photoprotective adaptation strategy against UVB damage, an increase of 58.9% phenolic compounds and 3.6% of carotenoids was observed. Overall, these results lead to the conclusion that both ultrastructural damage and observable changes in metabolism occurred in H. musciformis after only 3h of daily UVB exposure over a 7-d experimental period.

Effects of salinity on the growth rate, carrageenan yield, and cellular structure of Kappaphycus alvarezii (Rhodophyta, Gigartinales) cultured in vitro

Kappaphycus alvarezii was cultured in vitro under salinities ranging from 15 to 55 psu for 35 days to determine the differential effect on growth rate, carrageenan yield, and cellular structure. Plants kept in 15 psu died after 3 days, while plants cultured in 55 psu presented low growth rates during the entire experimental period (0.28% day−1). Plants cultured in 25, 35, and 45 psu showed growth rates normally associated with this species (between 3% and 4% day−1) and similar cellular morphology. Carrageenan yield was significantly higher in plants cultured in 25 psu in relation to the other treatments. As observed by light microscopy, plants cultured in 15 psu showed cellular turgidity and increased cell wall thickness, both consequences of hyposalinity. Chloroplasts and other membranous organelles underwent rupture and considerable disorganization in ultrastructure. Although branches from the 55 psu samples showed plasmolysis, cells were able to maintain chloroplast integrity, despite their rudimentary features. In high salinities, great concentrations of floridean starch grains were observed in subcortical cells, indicating their probable participation in osmoregulation. Based on these results, we defined the range of 25 to 45 psu as the limits of saline tolerance for K. alvarezii. While new field studies are required to confirm these results, it can be concluded that new sites, such as inactive or abandoned shrimp tanks with salinities up to 25 psu, could be considered for commercial farming.

Effects of UVB radiation on the agarophyte Gracilaria domingensis (Rhodophyta, Gracilariales): Changes in cell organization, growth and photosynthetic performance

The effects of ultraviolet radiation-B (UVBR) in apical segments of the red macroalgae Gracilaria domingensis (Kützing) Sonder ex Dickie were examined in vitro. Over a period of 21 days, the segments were cultivated and exposed to photosynthetically active radiation (PAR) at 80 μmol photons m −2 s −1 and PAR + UVBR at 1.6 W m −2 for 3 h per day. The samples were processed for electron microscopy, as well as histochemical analysis, and growth rate, photosynthetic pigment contents and photosynthetic performance were measured. Toluidine Blue reaction showed metachromatic granulations in vacuole and lenticular thickness, while Coomassie Brilliant Blue showed a higher concentration of cytoplasmic organelles, and Periodic Acid Schiff stain showed an increase in the number of floridean starch grains. UVBR also caused changes in the ultrastructure of cortical and subcortical cells, which included an increased number of plastoglobuli, changes in mitochondrial organization, destruction of chloroplast internal organization, and the disappearance of phycobilisomes. The algae cultivated under PAR-only showed growth rates of 6.0% day −1, while algae exposed to PAR + UVBR grew only 2.8% day −1. Compared with algae cultivated with PAR-only, the contents of photosynthetic pigments, including chlorophyll a, phycoerythrin, phycocyanin and allophycocyanin, decreased after exposure to PAR + UVBR, and significant differences were observed. Finally, analysis of these four photosynthetic parameters also showed reduction after exposure to PAR + UVBR: maximum photosynthetic rate, photosynthetic efficiency, photoinhibition and relative electron transport rate. Taken together, these findings strongly suggested that UVBR negatively affects the agarophyte G. domingensis.

Plastid development and floridean starch grain formation during carposporogenesis in the coralline red algaLithothrix aspergillum Gray

The development of the chloroplasts and the formation of floridean starch grains during carposporegenesis in the coralline algaLithothrix aspergillum Gray, was studied by electron microscopy. Proplastids are first recognized next to the plasmalemma in the youngest cell of the carposporangial filament.

THE ULTRASTRUCTURE OF CARPOSPOROGENESIS IN THE MARINE HEMIPARASITIC RED ALGAERYTHROCYSTIS SACCATA1

SUMMARYThe ultrastructure of carposporogenesis forErythrocystis saccatais described. The fusion and gonimoblast cells contain few organelles, and chloroplasts are in a proplastid state, with pit plugs between gonimoblast cells dissolving early in development. Carpospore development may be separated into 3 stages, the first stage being characterized by the appearance of straight‐profiled dictyosomes, fibrous vesicles, and an increase of discoid thylakoids within the chloroplasts. During the second, stage the dictyosomes assume a curved profile and striped vesicles are formed by the endoplasmic reticulum. The third stage is initiated by the disappearance of striped vesicles and the appearance of straight‐profiled dictyosomes secreting vesicles with cores. Mature carpospores consist of many cored vesicles, fibrous vesicles, and floridean starch grains. A single wall layer surrounds each carpospore since the carposporangial wall becomes incorporated into a mucilaginous matrix surrounding the spores.

ULTRASTRUCTURE OF THE CONCHOCELIS STAGE OF THE MARINE RED ALGA PORPHYRA LEUCOSTICTA1

SUMMARYThe ultrastructure of the Conchocelis or filamentous stage of Porphyra leucosticta was investigated. Each cell contains 1 or 2 parietal, stellate chloroplasts with a single pyrenoid in each chloroplast. The centrally located nucleus is irregularly shaped and contains 1–2 nucleoli. The cytoplasm has typical floridean starch grains and nonmernbrane‐bound lipid bodies. The cell wall is divided into an outer and an inner wall. Many lomasomes are associated with the cell membrane. Pit connections are found between cells, and their taxonomic significance is discussed.

CHROMATOPHORE DEVELOPMENT, PITS, AND OTHER FINE STRUCTURE IN THE RED ALGA, LOMENTARIA BAILEYANA (HARV.) FARLOW

Thin sections of the red alga, Lomentaria baileyana, a tubular member of the Rhodymeniales, were examined after permanganate fixation and Araldite embedding. Many of the cellular structures in Lomentaria were found to be similar to analogous structures in animals and higher plants. However, in the walls between cells are modified areas generally known as pits which are unique to the higher orders of red algae (Florideae). In this study the pits were found to consist of a plug-like structure surrounded by an uninterrupted membrane apparently continuous with the plasma membrane. Examination of the chromatophore revealed a characteristic limiting membrane, a relatively sparse distribution of plates, no grana, and a single disc apparently oriented parallel to the limiting membrane. In addition to their origin from non-lamellate proplastids, chromatophores were found capable of division by simple constriction. Floridean starch grains were observed outside the chromatophore and the possibility of an association of the first formed grains with portions of the endoplasmic reticulum is considered. Gland cells seem to have a high proportion of Golgi components (dictyosomes), and are believed to have some kind of secretory function. Many of the Golgi vesicles seem to open on the wall and presumably discharge their contents.