CELL-WALL FORMATION DURING THE CELL CYCLE OF PORPHYRIDIUM SP. (RHODOPHYTA)

The cell wall of the red microalgae Porphyridium sp. (UTEX 637) comprises a complex amorphous polysaccharide (6–7 × 10⁶ Da). The polysaccharide is made up of xylose, glucose, and galactose as the main sugars, as well as some minor sugars, protein, sulfate, and glucuronic acid, the latter two conferring a negative charge on the polysaccharide. In this study, we used synchronized cultures as one of the ways of unraveling the mechanism of biosynthesis of this complex polysaccharide by following cell-wall formation during the cell cycle. Synchronization of Porphyridium sp. was achieved with an alternating light:dark regime of 12:12 h LD and dilution of the culture at the end of the cycle. Under these conditions, cell duplication occurred between the 12th and 14th hours of the cycle. The following order of building toward formation of the final polysaccharide appeared to take place: Intermediate polysaccharides with molecular masses ranging from 0.5 × 10⁶ to 2 × 10⁶ Da appeared in succession during hours 2–6 of the cycle, and the full-sized polysaccharide was detected by the 8th hour. At the beginning of the cycle, xylose was the predominant sugar. Sulfur peaked at hours 2–4; glucose, galactose, and glucuronic acid at hours 8–12; and the minor sugars at hours 12–14. Upon incubation of low molecular mass polymer (0.5 × 10⁶ Da) collected from the 4th hour with cellular crude extract from cells of the 6th hour of the cycle, two intermediates were formed (0.8 × 10⁶ Da and 2 × 10⁶ Da). We suggest that the 0.5 × 10⁶ Da polymer intermediate, which is composed mainly of xylose, is the first polymer secreted into the medium, where it is further polymerized enzymatically to produce the 2 × 10⁶ Da polymer via an intermediate 0.8 × 10⁶ Da polymer. Later, the full-size polysaccharide is produced.     

A MODIFIED CELL WALL MUTANT OF THE RED MICROALGA RHODELLA RETICULATA RESISTANT TO THE HERBICIDE 2,6-DICHLOROBENZONITRILE1

The rigid component of the cell walls of red macroalgae, cellulose, is lacking in the red microalgae. Instead, the cells are encapsulated within an amorphous polysaccharide. These complex sul fated polysaccharides are composed of at least 10 different sugars, but their structure is not known, When the herbicide 2,6-dichlorobenzonitrile (DCB), a compound that specifically inhibits cellulose biosynthesis, was applied to cultures of the red microalga Rhodella reticulata upon inoculation, growth was inhibited. When added during the stationary phase of growth (after cell division had ceased), DCB did not affect cell number but it did inhibit polysaccharide production. A spontaneous mutant resistant to DCB was selected; it had physiological characteristics similar to those of the wild-type parent. The composition of the cell wall polysaccharide of the mutant was totally modified, being composed almost entirely (98% of its dry matter, as compared to 2.9% in the wild type) of methyl galactose, but retaining the same sulfate content. The molecular mass of the mutant polysaccharide was, however, similar to that of the wild-type parent (～6 × 10⁶ daltons), although its viscosity was significantly lower.     

Water-soluble polysaccharides of Opuntia ficus-indica cv “Burbank's Spineless”

Carbohydrate-containing polymers have been extracted with water from the fleshy, lobed stems of Opuntia ficus-indica cv “Burbank's Spineless”. By ion exchange chromatography, the material was separated into one neutral and two acidic fractions. Each fraction was separated in two by gel filtration. The neutral fractions consisted of two glucans and a glycoprotein, containing arabinose and galactose. All four acidic fractions contained galacturonic acid, arabinose, rhamnose, galactose and xylose in different proportions. The cell wall structure of O. ficus-indica is discussed.     

GLYCOPROTEIN MOIETY IN THE CELL WALL OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA) AS THE BIORECOGNITION SITE FOR THE CRYPTHECODINIUM COHNII-LIKE DINOFLAGELLATE

The Crypthecodinium cohnii -like heterotrophic dinoflagellate preys on the cells of the red microalga Porphyridium sp. UTEX 637, and not on other microalgae. The dinoflagellate contains enzymes that degrade the cell wall complex of this species of alga and not that of other red microalgae. The cells of the red microalgae are encapsulated within a cell wall complex composed of about 10 sugars, sulfate, and proteins. We previously hypothesized that the dinoflagellate recognizes the cell wall of this alga. In this study, we have shown that the biorecognition site is the 66-kDa glycoprotein in the algal cell wall complex. The methodology used in this study was based on changing the algal cell wall composition and examining the prey and chemosensory response of the dinoflagellate. The dinoflagellate was not attracted to the cell wall of other red microalgae, which are similar to that of Porphyridium sp., or to sugars composing its cell wall. However, the dinoflagellate preyed on and was attracted to Porphyridium sp. mutants (DCB resistant) having modified cell wall polysaccharide composition, probably because the 66-kDa cell wall glycoprotein was not changed. The dinoflagellate did not respond chemotactically to enzymatically degraded cell wall complex. Treatment of the cell wall complex with antiserum to the 66-kDa glycoprotein or with the lectin concanavalin A (con A), which binds specifically to α-d-mannosyl and α-d-glucosyl residues, did not affect the chemotactic attraction. However, prey by the dinoflagellate was prevented when the algal cells were blocked with antiserum specific to the 66-kDa glycoprotein or with con A. These latter results provide direct proof that the 66-kDa cell wall glycoprotein isthe recognition site and prey-prevention results from the blocking of this site on the cell wall.     

Water-soluble glycoproteins from Cannabis sativa (South Africa)

The water-soluble glycoproteins obtained from Cannabis leaves of plants grown from South African seeds have been further studied. Treatment of the glycoprotein fractions with NaOH in the presence of NaBH₄, resulted in a significant decrease in the serine content and a corresponding increase in alanine. The carbohydrate side chains released contained the sugar alcohol, galactitol. By treatment of the glycoprotein fractions with NaOH in the presence of Na₂SO₃, and subsequent acid hydrolysis, cysteic acid was formed. These data indicate that carbohydrate and protein are connected via serine-O-galactoside linkages. Further investigation of the structure of the carbohydrate part of the glycoproteins was carried out by methylation analysis, Smith-degradation and enzyme incubation. The present glycoprotein material of plants grown from South African seeds is similar to the material previously investigated, but in contrast to the latter, it is devoid of hexosamine.     

紫球藻及其多糖抗菌性能初探

本文报道紫球藻提取液及其多糖溶液抗病原细菌、真菌性能的研究结果。采用管碟法分别测定紫球藻培养分泌物、藻体破碎后的水提物和醇提物、胞外多糖和胞内多糖水溶液等的抗菌能力。结果表明,各样品均有一定抗菌能力,紫球藻水提物和胞外分泌物抗菌性能较强。经比较分析,多糖是抗菌作用的主要组分。紫球藻提取物及其多糖对革兰氏阳性细菌(特别是对金黄色葡萄球菌和八叠球菌)的抑菌作用较为明显,具有一定开发价值。     

LIGHT ACCLIMATION IN PORPHYRIDIUM PURPUREUM (RHODOPHYTA): GROWTH,PHOTOSYNTHESIS, AND PHYCOBILISOMES1

Acclimation to three photon flux densities (10, 35, 180 μE.m^?2.s^?1) was determined in laboratory cultures of Porphyridium purpureum Bory, Drew and Ross. Cultures grown at low, medium, and high PPFDs had compensation points of <3, 6, and 20 μE-m^?2.s^?1, respectively, and saturating irradiances in the initial log phase of 90, 115, 175 μE.m^?2.s^?1 and up to 240 μE.m^?2.s^?1 in late log phase. High light cells had the smallest photosynthetic unit size (phycobiliproteins plus chlorophyll), the highest photosynthetic capacity, and the highest growth rates. Photosystem I reaction centers (P700) per cell remained proportional to chlorophyll at ca. 110 chl / P700. However, phycobiliprotein content decreased as did the phycobilisome number (ca. 50%) in high light cells, where as the phycobilisome size remained the same as in medium and low light cells. We concluded that acclimation of this red alga to varied PPFDs was manifested by the plasticity of the photosystem II antennae with little, if any, effect noted on photosystem I.     

Polysaccharide from cell walls of Chlamydomonas reinhardtii

The cell wall fraction of the green alga Chlamydomonas reinhardtii contained about 25% carbohydrate after prolonged treatment with salivary amylase     

Cell wall glycoproteins from Chlamydomonas reinhardii are sulphated

Abstract Both the two major structural cell wall glycoproteins and the soluble excreted glycoproteins of Chlamydomonas reinhardii Levine WT II/32 contain low levels (approx. 1–4%) of sugar O-sulphate esters, asymmetrically distributed within the molecules. Preliminary characterization of their structure is described through [³⁵S] sulphate labelling experiments. The function of the sulphated glycoproteins is discussed in terms of their structural role and their water retaining properties.     

NOTE MOLECULAR ANALYSIS OF THE AHAS GENE OF PORPHYRIDIUM SP. (RHODOPHYTA) AND OF A MUTANT RESISTANT TO SULFOMETURON METHYL

Acetohydroxyacid synthase (AHAS) is the target enzyme of the sulfonylurea herbicides, and here we report the sequence of the gene from wild-type and herbicide-resistant Porphyridium sp. (Rhodophyta). The resistant mutant has a single residue substitution at a position known to confer herbicide resistance in E. coli and in plants. The rhodophyte gene is of cyanobacterial origin and distinct from the nuclear-encoded chlorophyte gene, which may be of mitochondrial origin.     

PROBING PHYCOBILISOME STRUCTURE BY IMMUNO-ELECTRON MICROSCOPY1

By immuno-electron microscopy it was shown that phycoerythrin is located on the outer surface of the phycobilisome and allophycocyanin is on the inside near the photosynthetic membrane in the red alga Porphyridium purpureum (Bory) Drew & Ross (P. cruentum). These findings are consistent with the idea that the phycobilisome junctions as a light harvesting antenna and energy sink, which directs the energy to chlorophyll in the photosynthetic membrane. A technique was devised in which unfixed phycobilisomes, attached to thylakoid vesicles, were separately reacted with three monospecific antisera (to B-phycoerythrin, R-phycocyanin and allophycocyanin) and the reaction products were secondarily marked by reaction with ferritin-conjugated goat-antirabbit gamma globulin fraction. This was subsequently followed by glutaraldehyde fixation and staining with phosphotungstic acid. The entire procedure was carried out on an electron microscope grid. The results confirm the previously proposed phycobilisome structural model.     

PROTOPLAST FUSION AND GENETIC COMPLEMENTATION OF PIGMENT MUTATIONS IN THE RED MICROALGA PORPHYRZDZUM SP.1

Protoplasts from two green pigment mutants of Porphyridium sp. (UTEX 637) containing a low phycoerythrin level were fused by exposure to polyethylene glycol (MW 6000) combined with a short heat shock (45° C, 5 min). Following regeneration on agar plates, red colonies arose in which complementation of the phycoerythrin deficiency had occurred. The complementation frequency was estimated to be 0.2%. Eight progeny showing red pigmentation were isolated and purified by consecutive transfers on agar plates. Characterization of the fusion progeny revealed that their phycobiliprotein and chlorophyll contents per cell were higher than those of their parental mutant strains and, in most strains, similar to that of the wild type. The fusion products proved to be stable over many growth cycles. The DNA content of the wild type and of the parental mutant strains was about 0.05 pg-cell^?1. Fusion progeny strains showed a variable DNA content: a few fusants contained the same amount of DNA as the wild type and the parental strains, while others had about 50% more DNA per cell. The DNA content of one of the progeny strains (CF1c) was double that of the wild type (0.1 pg. cell^?1). Cells of this fusion progeny contained one nucleus per cell, which suggests that nuclear fusion and the formation of a stable diploid followed cell fusion. Analysis of phycobilisome components of CF1c revealed complementation of linker polypeptides associated with phycoerythrin (γ subunits). CF1c contained, like the wild-type strain, four linker polypeptides; all of these were absent in one parental strain and one was absent in the second. To the best of our knowledge, this is the first report of protoplast fusion, formation of somatic hybrids, and the apparent completion of a parasexual cycle in a red microalga.     

A SPOROPHYTE CELL WALL PROTEIN OF THE RED ALGA PORPHYRA PURPUREA (RHODOPHYTA) IS A NOVEL MEMBER OF THE CHYMOTRYPSIN FAMILY OF SERINE PROTEASES1

A complementary DNA (cDNA) clone from a Porphyra purpurea (Roth) C. Agardh sporophyte-specific subtracted cDNA library was found to encode a protein similar to serine proteases of the chymotrypsin class. The encoded protein contains a typical signal peptide and is particularly similar to chymotrypsins in the regions surrounding the active site residues and the activation site where cleavage of the propeptide occurs. In addition, the six cysteine residues characteristic of chymotrypsins are conserved. However, two of the three residues of the active site His/Asp/Ser charge relay triad have been replaced, indicating that the protein is unlikely to have peptidase activity. Northern hybridization confirmed that this cDNA is derived from an abundant, sporophyte-specific messenger RNA (mRNA). The presence of signal peptide on the encoded protein and the abundance of its mRNA suggested that this protein might be localized in the cell wall. Consequently, sporophyte cell walls were isolated and a major protein having a molecular weight similar to that estimated for the encoded protein was purified. N-terminal sequence analysis indicated that this cell wall protein is identical to that encoded by the cDNA with the amino terminus of the mature protein beginning at the activation site. This cell wall structural protein appears to have evolved from a chymotrypsin-like progenitor but has been adapted to bind cell wall proteins and/or polysaccharides rather than to cleave proteins.