Effects of circadian rhythms of fluctuating temperature on growth and biochemical composition of <Emphasis Type="Italic">Ulva pertusa</Emphasis>

The marcoalga Ulva pertusa was cultured under (20 ± 2)°C, (20 ± 4)°C, (20 ± 6)°C, (20 ± 8)°C and (20 ± 10)°C circadian rhythms of fluctuating temperature conditions, and constant temperature of 20°C was used as the control. The growth rate of macroalga at (20 ± 2)°C, (20 ± 4)°C and (20 ± 6)°C were significantly higher than that at constant temperature of 20°C, while growth rate at (20 ± 8)°C and (20 ± 10)°C were significantly lower than that at constant temperature of 20°C. The growth rate of macroalga was a quadratic function of the thermal amplitude. Such a growth model can be described by G = β ₀ + β ₁(TA) + β ₂(TA)², where G represents the relative growth rate, TA is thermal amplitude in degree Celsius, β ₀ is the intercept on the G axis, and β ₁ and β ₂ are the regression coefficients. The optimal thermal amplitude for the growth of thallus at mean temperature of 20°C was estimated to be ± 3.69°C. Analysis of biochemical composition at the final stages of thaulls growth revealed that diel fluctuating temperature caused various influences (P < 0.05). The content of chlorophyll, protein and total solute carbohydrate at (20 ± 2)°C and (20 ± 4)°C were slightly higher than those at constant temperature of 20°C, however no statistically significant differences were found among them (P > 0.05). While osmolytes (total solute carbohydrate and free proline) at (20 ± 10)°C were significantly higher than that at 20°C (P < 0.05). Therefore, more chlorophyll and carbohydrate production might account for the enhancement in the growth of macroalga at the diel fluctuating temperatures in the present study. Handling editor: S. M. Thomaz     

New rhythmic changes in mitosis and growth in low differentiated green and red marine macroalgae

The cell division and vegetative growth of the thalli of simply differentiated macroalgae with a diffuse growth type—Ulva pseudocurvata (Chlorophyta) and Porphyra umbilicalis (Rhodophyta)-have been studied under natural and laboratory conditions. For this purpose the mitotic index and growth rate of algae were measured over 18 days. A diurnal rhythm of the mitotic index was revealed: the minimal mitotic index was registered in morning and daylight hours (for U. pseudocurvata 1–4%, for P. umbilicalis 0.5–2%), in the afternoon the index grew and reached its maximum 1 hour before dark (for U. pseudocurvata 12%, for P. umbilicalis 7%), then it slowly decreased during the night. In the studied algal species 2–3-and 6-day rhythms of mitotic index and growth rate were found for the first time both under natural and laboratory conditions. With constant white light these rhythms persisted for 9 days, this confirms the endogenous regulation of these rhythmic variations.     

Application of response surface method to evaluate the cytotoxic potency of Ulva fasciata Delile,a marine macro alga

Bioprospecting of marine natural products has recently produced a substantial number of drug candidates. Ulva fasciata Delile, belonging to the family Ulvaceae, is a green marine macro alga that grows profusely on the coastal seashore of South India. In the present study, we investigated the in vitro cytotoxic potential of a methanolic extract of U.fasciata Delile (MEUF) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay against human colon carcinoma (HT-29), human hepatocyte carcinoma (Hep-G2), and human breast carcinoma (MCF-7) cell lines. Response surface methodology (RSM) was applied using central-composite experimental design (CCD) to obtain optimum combined effect of concentration and cancer cells with highest cytotoxicity. The effect of concentration, cancer cell lines as independent variables on absorbance (OD), percent cell survival and percent cell inhibition as dependent variables was investigated. Maximum cytotoxic activity of MEUF was established for Hep-G2 with lowest OD or percent cell survival; highest percent cell inhibition with significant difference (p > 0.05) was compared to HT-29 and MCF-7.     

Effects of ocean acidification on growth and physiology of Ulva lactuca (Chlorophyta) in a rockpool‐scenario

Rising atmospheric CO₂‐concentrations will have severe consequences for a variety of biological processes. We investigated the responses of the green alga Ulva lactuca (Linnaeus) to rising CO₂‐concentrations in a rockpool scenario. U. lactuca was cultured under aeration with air containing either preindustrial pCO₂ (280 μatm) or the pCO₂ predicted by the end of the 21st century (700 μatm) for 31 days. We addressed the following question: Will elevated CO₂‐concentrations affect photosynthesis (net photosynthesis, maximum relative electron transport rate (rETR(max)), maximum quantum yield (Fv/Fm), pigment composition) and growth of U. lactuca in rockpools with limited water exchange? Two phases of the experiment were distinguished: In the initial phase (day 1–4) the Seawater Carbonate System (SWCS) of the culture medium could be adjusted to the selected atmospheric pCO₂ condition by continuous aeration with target pCO₂ values. In the second phase (day 4–31) the SWCS was largely determined by the metabolism of the growing U. lactuca biomass. In the initial phase, Fv/Fm and rETR(max) were only slightly elevated at high CO₂‐concentrations, whereas growth was significantly enhanced. After 31 days the Chl a content of the thalli was significantly lower under future conditions and the photosynthesis of thalli grown under preindustrial conditions was not dependent on external carbonic anhydrase. Biomass increased significantly at high CO₂‐concentrations. At low CO₂‐concentrations most adult thalli disintegrated between day 14 and 21, whereas at high CO₂‐concentrations most thalli remained integer until day 31. Thallus disintegration at low CO₂‐concentrations was mirrored by a drastic decline in seawater dissolved inorganic carbon and HCO₃^?. Accordingly, the SWCS differed significantly between the treatments. Our results indicated a slight enhancement of photosynthetic performance and significantly elevated growth of U. lactuca at future CO₂‐concentrations. The accelerated thallus disintegration at high CO₂‐concentrations under conditions of limited water exchange indicates additional CO₂ effects on the life cycle of U. lactuca when living in rockpools.     

ANALYSIS OF EXPRESSED SEQUENCE TAGS FROM THE GREEN ALGA ULVA LINZA (CHLOROPHYTA)1

There is a general lack of genomic information available for chlorophyte seaweed genera such as Ulva, and in particular there is no information concerning the genes that contribute to adhesion and cell wall biosynthesis for this organism. Partial sequencing of cDNA libraries to generate expressed sequence tags (ESTs) is an effective means of gene discovery and characterization of expression patterns. In this study, a cDNA library was created from sporulating tissue of Ulva linza L. Initially, 650 ESTs were randomly selected from a cDNA library and sequenced from their 5′ ends to obtain an indication of the level of redundancy of the library (21%). The library was normalized to enrich for rarer sequences, and a further 1920 ESTs were sequenced. These sequences were subjected to contig assembly that resulted in a unigene set of approximately 1104 ESTs. Forty‐eight percent of these sequences exhibited significant similarity to sequences in the databases. Phylogenetic comparisons are made between selected sequences with similarity in the databases to proteins involved in aspects of extracellular matrix/cell wall assembly and adhesion.     

Isolation of Pollutant (Pine Needle Ash)-Responding Genes from Tissues of the Seaweed Ulva Pertusa

Genetic responses of the seaweed Ulva pertusa to pine needle ash have been compared using differential display technique. The tissue viability was assessed to evaluate the stress level with triphenyltetrazolium chloride. Total RNA, from tissues treated in seawater containing ash, was reverse transcribed and amplified by PCR with arbitrary primers. The genetic fragments responding to the stress were selectively isolated from agarose gel and sequenced with a DNA auto sequencer. According to sequence analysis, an ash-inducible gene (342 bp) and an ash-suppressed gene (1690 bp) were identified as hypothetical proteins.     

ASYMMETRY OF EYESPOT AND MATING STRUCTURE POSITIONS IN ULVA COMPRESSA (ULVALES,CHLOROPHYTA) REVEALED BY A NEW FIELD EMISSION SCANNING ELECTRON MICROSCOPY METHOD1

Gametes of the marine green alga Ulva compressa L. are biflagellate and pear shaped, with one eyespot at the posterior end of the cell. The species is at an early evolutionary stage between isogamy and anisogamy. In the past, zygote formation of green algae was categorized solely by the relative sizes of gametes produced by two mating types (+ and ?). Recently, however, locations of cell fusion sites and/or mating structures of gametes have been observed to differ between mating types in several green algae (asymmetry of cell fusion site and/or mating structure positions). To use this asymmetry for determining gamete mating type, we explored a new method, field emission scanning electron microscopy (FE‐SEM), for visualizing the mating structure of U. compressa. When gametes were subjected to drying stress in the process of a conventional critical‐point‐drying method, a round structure was observed on the cell surfaces. In the mating type MGEC‐1 (mt⁺), this structure was located on the same side of the cell as the eyespot, whereas it was on the side opposite the eyespot in the mating type MGEC‐2 (mt^?). The gametes fuse at the round structures. TEM showed an alignment of vesicles inside the cytoplasm directly below the round structures, which are indeed the mating structures. Serial sectioning and three‐dimensional construction of TEM micrographs confirmed the association of the mating structure with flagellar roots. The mating structure was associated with 1d root in the MGEC‐1 gamete but with 2d root in the MGEC‐2 gamete.     

Isolation of protoplasts and ion channel recording of plasma membrane patches and whole-cell recordings of the marine alga, <Emphasis Type="Italic">Ulva pertusa</Emphasis> (Chlorophyta)

Whole-cell patch-clamp techniques were used to study ion channels of a marine alga. High quality protoplasts suitable for electrophysiological studies were isolated from the green marine alga, Ulva pertusa, using enzyme mixtures consisting of cellulase and abalone power and identified by calcofluor fluorescence. The vitality of protoplasts varied depending on the alga growth stage, and those isolated from younger tissue in March maintained a high vitality with high sealing success rate compared with protoplasts isolated from mature or non-growing plants in August or November. In the whole-cell configuration, large inward currents were elicited by negative voltage pulses. The voltage-dependent component was predominantly carried by Cl⁻, as confirmed by the use of the Cl⁻ channel inhibitor DIDS and reversal potential of current-voltage plots. This evidence suggests that hyperpolarization-activated Cl⁻ permeable channels are responsible for the influx of Cl⁻ into U. pertusa cells. Voltage-dependent outward currents were also recorded in several protoplasts, and their properties need further investigation.     

DEVELOPMENT OF PROTOPLASTS OF ULVA FASCIATA (CHLOROPHYTA) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate protoplasts of the green alga Ulva fasciata Delile and subsequently induce them to form a microthallus suspension for algal seed stock. The protoplasts were covered with secreted mucilage following 6 h of culture when viewed with SEM. The mucilage fused to form thick layers during day 1 of culture. Microfibrillar cell walls were deposited into the thick layers of mucilage on the 5th day of culture. An average of about 10% of the freshly isolated protoplasts began to divide at 6–14 days. These protoplasts subsequently developed varied morphologies, depending on the time of collection during the year. Protoplasts isolated from U. fasciata collected in March to June developed frond thalli or microthalli when they were cultured in low or high densities (cells/area), respectively. The microthallus suspension was cultured for more than two years at 10–40 μ mol·m^{− 2} ·s^{− 1} . Frond thalli formed when the suspension was cultivated at 100–160 μ mol·m^{− 2} ·s^{− 1} . Therefore, microthallus suspension can serve as a seed stock of U. fasciata .     

UV‐B INDUCTION OF UV‐B PROTECTION IN ULVA PERTUSA (CHLOROPHYTA)1

The green macroalga Ulva pertusa Kjellman produced UV‐B absorbing compounds with a prominent absorption maximum at 294 nm in response only to UV‐B, and the amounts induced were proportional to the UV‐B doses. Under a 12:12‐h light:dark regime, the production of UV‐absorbing compounds occurred only during the exposure periods with little turnover in the dark. There was significant reduction in growth in parallel with the production of UV‐B absorbing compounds. The polychromatic action spectrum for the induction of UV‐B absorbing compounds in U. pertusa exhibits a major peak at 292 nm with a smaller peak at 311.5 nm. No significant induction was detected above 354.5 nm, and radiation below 285 nm caused significant reduction in the levels of UV‐B absorbing compounds. After UV‐B irradiation at 1.0 W·m^?2 for 9 h, the optimal photosynthetic quantum yield of the samples with UV‐B absorbing compounds slightly increased relative to the initial value, whereas that of thalli lacking the compounds declined to 30%–34% of the initial followed by subsequent recovery in dim light of up to 84%–85% of the initial value. There was a positive and significant relationship between the amount of UV‐B absorbing compounds with antioxidant activity as determined by the α,α‐diphenyl‐β‐picrylhydrazyl scavenging assay. In addition to mat‐forming characteristics and light‐driven photorepair, the existence and antioxidant capacity of UV‐B absorbing compounds may confer U. pertusa a greater selective advantage over other macroalgae, thereby enabling them to thrive in the presence of intense UV‐B radiation.