DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts

The effects of methylation on plant viral DNA replication have been studied inNicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m⁵C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m⁵C residues were substituted for cytosine residuesin vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequencesin vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.     

DEVELOPMENT OF PROTOPLASTS FROM HOLDFASTS AND VEGETATIVE THALLI OF MONOSTROMA LATISSIMUM (CHLOROPHYTA, MONOSTROMATACAE) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate the protoplasts of the green alga Monostroma latissimum Wittrock and subsequently induce them to form algal filaments to act as an algal "seed" stock. Protoplasts of the alga were isolated enzymatically with 4% cellulase Onozuka R-10 and 2% Macerozyme R-10. The highest number of protoplasts was obtained on a 50-rpm shaker with 1.2 M of sorbitol after 6 h of incubation, with a yield of 9 × 106 protoplasts·g−1 of fresh thallus (including holdfast). Protoplasts from both holdfasts and erect thalli usually began to form new cell walls within 5 h after isolation and began to divide from day 6 to day 9 in PES medium; cell clusters, filaments, and/or tubular thalli were formed from day 14 to day 18. For algae collected in March, about 60% of protoplasts isolated from vegetative thalli regenerated to form tubular thalli, and about 45% of protoplasts isolated from holdfasts regenerated to form filaments. However, for algae collected in May, about 1% of protoplasts isolated from vegetative thalli developed directly to form tubular thalli, and 59% of protoplasts regenerated to form cell clusters without the ability to differentiate, whereas protoplasts isolated from holdfasts failed to develop. Regenerated filaments were kept in an incubator for more than 3 years at 24° C under the low irradiance of 66μmol photons·m−2·s−1. After this time, they retained the ability to develop to form tubular thalli under irradiance of 166 and 300 μmol photons·m−2·s−1 at 18°–30° C. Subsequently, these tubular thalli can develop to form leafy thalli after being cultivated at high irradiance of 300 μmol photons·m−2·s−1 and at 18°–22° C. Therefore, the filaments could serve as"seed" stock for algal mass culture.     

Protoplast isolation optimization and regeneration of cell wall in Gracilaria gracilis (Gracilariales, Rhodophyta)

This paper reports the first successful isolation and cell wall regeneration of Gracilaria gracilis (Stackhouse) Steentoft, Irvine et Farnham protoplasts. These results form an important foundation for the development of a successful tissue culture system for G. gracilis. Initially, an isolation protocol was optimized by investigation of the effects of the enzyme constituents and concentrations, the pre-treatment of thalli, the incubation period and temperature, and the pH of the enzymatic medium on protoplast yields. A pre-treatment of G. gracilis thalli with 1 % (w/v) papain for 30 min followed by a 3-h enzymatic digestion of thalli with an enzymatic mixture containing 2 % (w/v) cellulase Onozuka R-10, 1 % (w/v) macerozyme R-10, and 10 U mL^?1 agarase at pH 6.15 was found to produce the highest yield of protoplasts at 22 °C. Reliably high yields (20–30?×?10⁵ protoplasts g^?1 f.wt) of protoplasts could be obtained from G. gracilis thalli when this optimized protocol was used. Cell wall re-synthesis by G. gracilis protoplasts, which constitutes the first step towards whole plant regeneration, was followed using calcoflour staining and scanning electron microscopy. Protoplasts were shown to complete the initial stages of cell wall re-synthesis within the first 24 h of culturing.     

Heat stress and the photosynthetic electron transport chain of the lichen <Emphasis Type="Italic">Parmelina tiliacea</Emphasis> (Hoffm.) Ach. in the dry and the wet state: differences and similarities with the heat stress response of higher plants

Thalli of the foliose lichen species Parmelina tiliacea were studied to determine responses of the photosynthetic apparatus to high temperatures in the dry and wet state. The speed with which dry thalli were activated by water following a 24 h exposure at different temperatures decreased as the temperature was increased. But even following a 24 h exposure to 50°C the fluorescence induction kinetics OJIP reflecting the reduction kinetics of the photosynthetic electron transport chain had completely recovered within 128 min. Exposure of dry thalli to 50°C for 24 h did not induce a K-peak in the fluorescence rise suggesting that the oxygen evolving complex had remained intact. This contrasted strongly with wet thalli were submergence for 40 s in water of 45°C inactivated most of the photosystem II reaction centres. In wet thalli, following the destruction of the Mn-cluster, the donation rate to photosystem II by alternative donors (e.g. ascorbate) was lower than in higher plants. This is associated with the near absence of a secondary rise peak (~1 s) normally observed in higher plants. Analysing the 820 nm and prompt fluorescence transients suggested that the M-peak (occurs around 2–5 s) in heat-treated wet lichen thalli is related to cyclic electron transport around photosystem I. Normally, heat stress in lichen thalli leads to desiccation and as consequence lichens may lack the heat-stress-tolerance-increasing mechanisms observed in higher plants. Wet lichen thalli may, therefore, represent an attractive reference system for the evaluation of processes related with heat stress in higher plants.     

Plant regeneration from protoplasts of Sphacelaria (Phaeophyceae)

Protoplast were isolated from a filamentous brown alga, Sphacelaria sp. (Sphacelariales, Phaeophyta), using alginate-lyases extracted from marine molluscs, and commercial pectinase and cellulase. Yields were about 4000 protoplasts per gram of fresh tissue. Different types of protoplasts, originating from apical, subapical, nodal and internodal cells, could be readily identified based on their size and pigmentation. Apical cells produced a higher percentage of protoplasts (approx. 2%), compared with other cell types. All apical-cell protoplasts regenerated into new thalli and most other types of protoplasts divided at least once in culture, but did not develop further.     

Detection of epigenetic variation in tissue-culture-derived plants of <Emphasis Type="Italic">Doritaenopsis</Emphasis> by methylation-sensitive amplification polymorphism (MSAP) analysis

Somaclones exhibiting variations with flower characteristics were recovered from the tissue-culture-derived plants of Doritaenopsis. Two molecular techniques, random amplified polymorphic DNA (RAPD) and methylation-sensitive amplification polymorphism (MSAP) analyses, were used to characterize the somaclones. RAPD analysis, using 100 randomly selected primers, failed to differentiate variants and normal plants, even though some primers (six out of 100 primers) exhibited 6–10 distinct banding patterns. However, MSAP analysis revealed the differences in the DNA methylation patterns in the normal and variant plants which were correlated with phenotypic variation. In all, 311, 337, 366, and 343 fragments were obtained with normal and V1, V2, and V3 variant plants, respectively; each representing recognition site cleaved by either or both of the isoshizomers were amplified using 12 combination of primers. A total of 36 (11.6%), 77 (22.9%), 73 (19.9%), and 47 (13.7%) sites were found to be methylated at cytosine in the genomes of normal and V1, V2, and V3 variant Doritaenopsis plants. This study demonstrates usefulness of MSAP to detect DNA methylation events in tissue cultured Doritaenopsis plants.     

Production of bioflavor by regeneration from protoplasts of Ulva pertusa (Ulvales,Chlorophyta)

Protoplasts were isolated from thalli of Ulva pertusa using a mixed enzyme solution of 2.0% Cellulase Onozuka R-10, 2.0% Macerozyme R-10, and 2.0% Driselase. Isolated protoplasts regenerated cell walls, developed into thalli, and propagated in large numbers under aeration in the preparative scale-culture system. Typical bioflavor compounds produced from the regenerated plants, as well as from field-collected plants, were found to be long chain aldehydes, which gave a typical seaweed odor. The long chain aldehydes were formed enzymatically from unsaturated fatty acids and released into the culture fluid. A Percoll/mannitol discontinuous density gradient separation of the heterogeneous protoplasts led to a selection of cell lines with high production of bioflavor. The cells that regenerated from protoplasts were immobilized by polymer matrices such as alginate, -carrageenan, agarose, and agar. Living cells entrapped in alginate beads in aerated cultures survived best. However, the beads started to breakdown after two months. The immobilized cells demonstrated a higher bioflavor production than did the cultured cells.     

Characterization of the level,target sites and inheritance of cytosine methylation in tomato nuclear DNA

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mCT. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.     

A SIMPLE METHOD FOR ISOLATING FILAMENTS AS “ALGAL SEED STOCK” FROM MONOSTROMA LATISSIMUM (CHLOROPHYTA) GERMLINGS,AND APPLICATION FOR MASS CULTIVATION1

Germlings were grown from Monostroma latissimum Wittr. reproductive cells on nylon ropes. Holdfast threads and some uniseriate filaments were observed to have penetrated the fibers of the dispersed ropes. The algal filaments were easily isolated and prepared for cultivation, in comparison to the methods of enzymatically isolated algal protoplasts. Under low light (60–100 μmol photons · m^?2 · s^?1), the algal filaments grew to form a filamentous mass. When cultivated under stronger light (300–600 μmol photons · m^?2 · s^?1), they grew to initially form tubular thalli and then, when cultivated under light intensities >700 μmol photons · m^?2 · s^?1, formed foliaceous thalli. Consequently, the filaments were homogenized into small sections and then sewed on the nylon rope for algal mass cultivation. Under high‐intensity natural light, they grew to form leafy thalli.     

PHYSIOLOGICAL COMPARISON OF THE ISOMORPHIC LIFE HISTORY PHASES OF THE HIGH INTERTIDAL ALGA ENDOCLADIA MURICATA (RHODOPHYTA)1

The isomorphic phases of Endocladia muricata (Post. & Rupr.) J. Ag. Were compared for photosynthetic and respiratory difference in response to a variety of environmental manipulations. Photosynthetic light response during submergence at 15° C and the pattern of respiratory recovery following prolonged emergence (3 h) at either 15° or 30° C were similar between gametophytes and tetrasporophytes. The phases showed the same ability to photosynthesize and respire during emergence at each temperature tested (15°, 25°, and 35° C, fully hydrated thalli) and at various desiccation state (measured at 25° C only). Submerged rates of photosynthesis following prolonged emergence at 15° and 30° C were, however slightly greater (17%) for tetrasporophytes as compared to gametophytes. Regardless of the life history phase, plants incubated at 15° C during emergence recovered more completely than plants incubated at 30° C. Photosynthetic recovery after 1 h in plants incubated at 15° C often “spiked” and yielded rates as great as 185% of pretreatment rates. Increased photosynthetic rates during recovery were absent for the 30° C incubations. The initial photosynthetic recovery of plants collected from the upper limits of distribution was greater than that of plants collected from the lower limits. Recovered rates of respiration were highly variable over time. Respiration often exceeded pretreatment values more then threefold, and the elevated rates were sustained for 12 h. Photosynthesis and respiration in air were comparable to rates in seawater and varied slightly with increasing temperature. Photosynthetic and respiratory rates also decreased with increasing tissue water loss. Thus, only slight differences in physiological performance were observed between phases and individuals collected from different vertical positions. Metabolic differences were transient and apparent only under experimental conditions that modeled extreme environmental conditions.     

Life histories of Colpomenia sinuosa and Hydroclathrus clathratus (Scytosiphonaceae, Phaeophyceae) in culture

Life-history studies in culture were carried out on Colpomenia sinuosa (Mertens ex Roth) Derbès et Solier and Hydroclathrus clathratus (C. Agardh) Howe (Scytosiphonales, Phaeophyceae) from Japan. These species showed a heteromorphic life-history pattern with an alternation between erect thalli bearing plurilocular zoidangia and prostrate thalli bearing ectocarpoid plurilocular and unilocular zoidangia. Plurizoids released from erect and prostrate thalli developed into prostrate thalli. Unizoids, however, developed into erect thalli. Prostrate thalli produced plurilocular zoidangia in long-day conditions and unilocular zoidangia in short-day conditions at 10-20°C. Prostrate thalli of C. sinuosa formed ascocysts. Germlings of both species did not grow at 5°C.     

Bisprimer--a program for the design of primers for bisulfite-based genomic sequencing of both plant and Mammalian DNA samples

Plants and animals differ in the sequence context of the methylated sites in DNA. Plants exhibit cytosine methylation in CG, CHG, and CHH sites, whereas CG methylation is the only form present in mammals (with an exception of the early embryonic development). This fact must be taken into account in the design of primers for bisulfite-based genomic sequencing because CHG and CHH sites can remain unmodified. Surprisingly, no user-friendly primer design program is publicly available that could be used to design primers in plants and to simultaneously check the properties of primers such as the potential for primer-dimer formation. For studies concentrating on particular DNA loci, the correct design of primers is crucial. The program, called BisPrimer, includes 2 different subprograms for the primer design, the first one for mammals and the second one for angiosperm plants. Each subprogram is divided into 2 variants. The first variant serves to design primers that preferentially bind to the bisulfite-modified primer-binding sites (C to U conversion). This type of primer preferentially amplifies the bisulfite-converted DNA strands. This feature can help to avoid problems connected with an incomplete bisulfite modification that can sometimes occur for technical reasons. The second variant is intended for the analysis of samples that are supposed to consist of a mixture of DNA molecules that have different levels of cytosine methylation (e.g., pollen DNA). In this case, the aim is to minimize the selection in favor of either less methylated or more methylated molecules.     

Regeneration of plantlets fromEnteromorpha (Ulvales,Chlorophyta) protoplasts in axenic culture

High yields of protoplasts have been obtained from vegetative thalli of three species ofEnteromorpha by enzymatic degradation of the cell wall. Several commercial and crude enzymes prepared from the digestive system and hepatopancrease of abalone and top-shell were tested at different concentrations and combinations to evaluate the yield. Commercial enzymes in combination with either abalone or top-shell crude enzymes, consistently produced a high yield of protoplasts from all three species. High regeneration rate (85–95%) occurred in the protoplasts cultured at a density greater than 1.72 × 10³ cells cm⁻² at 20 and 25°C. Light intensities tested in the present study did not affect protoplast wall formation and regeneration. Protoplasts, after regenerating the cell wall, followed different types of developmental patterns under identical culture conditions. In one type some cells underwent repeated cell divisions and formed a round and oval shaped hollow thallus with a single layer of cells. In the second type many cells underwent one or two cell divisions (occasionally no division) and soon matured and discharged many motile spores, which on germination grew into normal plantlets. In the third type some cells divided irregularly to form a mass of callus-like cells (exceptE. prolifera). Culture medium supplemented with either mannitol, sorbitol, dextrose, saccharose or NaCl at higher concentrations (> 0.4 M) inhibited cell division and further differentiation in all species. author for correspondence     

THE LIFE CYCLE OE SARGASSUM HORNERI (PHAEOPHYTA) IN LABORATORY CULTURE1

The life cycle of the large dioecious alga Sargassum horneri (Turner) C. Agardh was completed in unialgal culture by controlling photoperiod in relation to the phase of growth. Embryos isolated from a naturally grown female thallus gave rise to early germlings that rapidly formed blades under both short-day (9 h L) and long-day (15 h L) conditions at 20° C Shoot elongation, which followed early blade formation, occurred under the short-day conditions hut not under the long-day conditions. Functional female and male receptacles developed when thalli 8–14 cm long grown under the short-day conditions were transferred to the long-day conditions; gamete fusion occurred when male and female thalli were grown together. Fertilized oospores gave rise to normal thalli in a manner similar to that for in situ plants. Thus, the life cycle of S. horneri was completed in laboratory culture.     

General introduction to the lists of threatened biotopes,flora and fauna of the trilateral Wadden Sea Area (red data book)

The effect of the acclimation temperature on the temperature tolerance ofPorphyra leucosticta, and on the temperature requirements for growth and survival ofEnteromorpha linza was determined under laboratory conditions. Thalli ofP. leucosticta (blade or Conchocelis phases), acclimated to twenty-five degrees, survived up to 30°C, i.e. 2°C more than those acclimated to 15°C which survived up to 28°C. Lower temperature tolerance of bothPorphyra phases that were acclimated to 15°C was −1°C after an 8-week exposure time at the experimental temperatures. The upper temperature tolerance ofE. linza also increased by 2°C, i.e. from 31 to 33°C, when it was acclimated to 30°C instead of 15°C. The lower temperature tolerance increased from 1 to −1°C, when it was acclimated to 5°C instead of 15°C.E. linza thalli acclimated for 4 weeks to 5 or 10°C reached their maximum growth at 15°C, i.e. at a 5°C lower temperature than those acclimated to 15 or 30°C. These thalli achieved higher growth rates in percent of maximal growth at low temperatures than those acclimated to 15 or 30°C. Thalli acclimated for 1 week to 5°C reached their maximum growth rate at 20°C and achieved growth rates at low temperatures similar to those recorded for thalli acclimated to 15°C. Thalli ofE. linza acclimated for 4 weeks to 5°C lost this acclimation after being post-cultivated for the same period at 15°C. That was not the case with thalli acclimated for 8 weeks to 5°C and post-acclimated for 4 weeks to 15°C. These thalli displayed similar growth patterns at 10–25°C, while a decline of growth rate was observed at 5 or 30°C. The significance of the acclimation potential ofE. linza with regard to its seasonality in the Gulf of Thessaloniki, and its distribution in the N Atlantic, is also discussed.     

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 .