Physiological and Biochemical Role of Brassinosteroids and Their Structure-Activity Relationship in the Green Alga Chlorella vulgaris Beijerinck (Chlorophyceae)

This paper studies the influence of the 7-oxalactone type of brassinosteroids (BRs) and 6-ketone upon the biological activity of the alga Chlorella vulgaris (Chlorophyceae). The results of the study indicate significant differences in the growth and metabolism of C. vulgaris cells caused by the different chemical structures of the BRs used. The most significant differences in the stimulation of the growth of the biomass and metabolites contained in it were caused by structural differences in the B ring of BRs. It was found that in C. vulgaris 7-oxalactone type of BRs [brassinolide (BL) and its derivatives] are more active than 6-ketone type of BRs [castasterone (CS) and its derivatives]. It was found that BRs used within the range of concentration of 10⁻¹² to 10⁻⁸ m stimulate two- to threefold the growth and division of C. vulgaris cells. The most stimulating influence upon the number of the algal cells and the phosphorus, chlorophyll, and monosaccharides contained in the alga, as well as the intensity of the photosynthesis, and sugar and glycolate excretion was demonstrated by BL at a concentration 10⁻⁸ m in the 36th h of cultivation. HomoCS was characterized by the lowest biological activity. In turn, after the 48th h an inhibition of the rate of growth and development of the alga takes place. In the range from 10⁻⁷ to 10⁻⁶ m the inhibition of growth and development of the alga was manifested by BRs. During the further toxic activity of BRs the cells of C. vulgaris undergo complete degradation. In turn, in concentrations lower than 10⁻¹² m, BRs do not exert any biologically significant influence upon C. vulgaris cells. On the basis of the study, the biological activity of BRs was arranged in the following order: BL > 24-epiBL > homoBL > CS > 24-epiCS > homoCS. Received July 21, 1997; accepted April 7, 1998     

大型海藻龙须菜抽提液对褶皱臂尾轮虫生命表参数的影响

大型海藻富含多种活性物质,具有抗衰老等生物活性;轮虫是良好的潜在抗衰老研究模式生物。本研究以褶皱臂尾轮虫(Brachionus plicatilis)作为实验对象,研究了不同浓度的大型海藻龙须菜抽提液(0,250,500,750,1000 mg/L)和不同浓度的食物(蛋白核小球藻和普通小球藻)对褶皱臂尾轮虫生命表参数的影响。结果表明:与对照组相比,食物浓度为1.0×10~6个/mL蛋白核小球藻时,不同浓度龙须菜抽提液对轮虫产卵数、平均寿命、净生长率以及世代时间有显著促进效应(P0.05);轮虫平均产卵数及寿命在龙须菜抽提液浓度750 mg/L处达到最高,分别为16只和13.9d(P0.05)。食物浓度为2.0×10~6个/mL普通小球藻时,轮虫平均产卵数和寿命在抽提液浓度为500 mg/L处达到最高,分别为16只和13.6d(P0.05),轮虫平均寿命和净生长率均有显著提高(P0.05)。相同龙须菜抽提液浓度下,食物浓度为1.0×10~6个/mL蛋白核小球藻下轮虫的净生长率、世代时间均显著高于食物浓度为2.0×10~6个/mL蛋白核小球藻培养的轮虫(P0.05);食物浓度为2.0×10~6个/mL时,普通小球藻培养轮虫的净生长率和世代时间均显著高于蛋白核小球藻实验组(P0.05)。交互作用分析显示,龙须菜抽提液与小球藻的交互作用对褶皱臂尾轮虫的内禀增长率有显著影响(P0.05)。研究结果表明,大型海藻龙须菜抽提液对褶皱臂尾轮虫的生长与生殖有促进作用,延长轮虫寿命。     

Effects of brassinazole,an inhibitor of brassinosteroid biosynthesis,on light- and dark-grown<Emphasis Type="Italic"> Chlorella vulgaris</Emphasis>

Treatment of cultured Chlorella vulgaris Beijerinck cells with 0.1–10 M brassinazole (Brz2001), an inhibitor of brassinosteroid (BR) biosynthesis, inhibits their growth during the first 48 h of cultivation in the light. This inhibition is prevented by the co-application of BR. This result suggests that the presence of endogenous BRs during the initial steps of the C. vulgaris cell cycle is indispensable for their normal growth in the light. In darkness, a treatment with 10 nM brassinolide (BL) promotes growth through the first 24 h of culture, but during the following 24 h the cells undergo complete stagnation. Treatment of dark-grown cells with either Brz2001 alone, or a mixture of 10 nM BL and 0.1/10 M Brz2001, also stimulates their growth. The effects of treatment with 10 nM BL mixed with 0.1–10 M of a mevalonate-pathway inhibitor (mevinolin), or a non-mevalonate-pathway inhibitor (clomazone), were also investigated. Mevinolin at these concentrations did not inhibit growth of C. vulgaris; however, clomazone did. Addition of BL overcame the inhibition. These results suggest that the mevalonate pathway does not function in C. vulgaris, and that the non-mevalonate pathway for isopentenyl diphosphate biosynthesis is responsible for the synthesis of one of the primary precursors in BR biosynthesis.Abbreviations Brz Brassinazole - BL Brassinolide - BR Brassinosteroid - Clo Clomazone - DMAPP Dimethylallyl diphosphate - IPP Isopentenyl diphosphate - MVA Mevalonic acid - Mev Mevinoline     

Two-Component Signal Transduction Systems of Desulfovibrio vulgaris: Structural and Phylogenetic Analysis and Deduction of Putative Cognate Pairs

A large number of two-component signal transduction systems (TCSTS) including 59 putative sensory histidine kinases (HK) and 55 response regulators (RR) were identified from the Desulfovibrio vulgaris genome. In this study, the structural and phylogenetic analyses of all putative TCSTSs in D. vulgaris were performed. The results showed that D. vulgaris contained 21 hybrid-type HKs, implying that multiple-step phosphorelay may be a common signal transduction mechanism in D. vulgaris. Despite the low sequence similarity that restricted the resolution of the phylogenetic analyses, most TCSTS components of D. vulgaris were found clustered into several subfamilies previously recognized in Escherichia coli and Bacillus subtilis. An attempt was made in this investigation to identify the possible cognate HK-RR pairs not linked on the chromosome in D. vulgaris based on similar expression patterns in response to various environmental and genetic changes. Expression levels of all HK and RR genes were measured using whole-genome microarrays. Five groups of HK-RR genes not linked on the chromosome were identified as possible cognate pairs in D. vulgaris. The results provided a preliminary list of possible cognate HK-RR pairs and constitute a basis for further exploration of interaction and physiological function of TCSTSs in D. vulgaris. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Gail Simmons]     

Harvesting of Chlorella vulgaris using a bioflocculant from Paenibacillus sp. AM49

Microbial flocculants for harvesting mass cultured Chlorella vulgaris were screened and that from Paenibacillussp. AM49 was identified as the best. The flocculation efficiency of this bioflocculant increased with the pH within a range of pH 5–11 and was 83%, which was higher than the 72% and 78% produced by aluminum sulfate and polyacrylamide, respectively. The highest flocculation efficiency was with 6.8 mm CaCl₂ as co-flocculant. The bioflocculant from Paenibacillussp. AM49 can be used effectively to harvest C. vulgaris from large-scale cultures.     

Chemical,physical and biological changes associated with chara succession in farm ponds

Four farm ponds in various stages of Chara vulgaris succession -from the initial invasion of the alga to its nearly complete replacement-were followed each week from ice cover to ice cover. Chemical, physical, and biological parameters were analyzed in order to determine differences between ponds and to discover the biological and physicochemical factors associated with succession in Chara vulgaris ponds. The data were analyzed statistically by intraclass (single factor) analysis of variance, the Student's t-test, and stepwise multiple regression analysis.The initial invasion of C. vulgaris appeared to be preceded by a lowering of the nutrient levels of the pond by Zygnematales growth and by the substrate-stabilizing growth of Rhizoclonium sp. Chara-dominated ponds had significantly lower free CO₂, bicarbonate alkalinity, and total hardness readings. Carbonate alkalinity, low phytoplankton productivity, high dissolved O₂, low phosphate, and high Secchi disk readings were also characteristic of ponds in which C. vulgaris was dominant.In ponds where C. vulgaris appeared in the process of being replaced, the physicochemical factors were more closely associated with phytoplankton productivity and diversity and less closely with submerged vegetation species.Succession from C. vulgaris domination to a Najas-Potamogeton community appeared to involve shading of the former by the latter and was accelerated by the smothering effect of decaying debris caused by Gloeotrichia sp.In the final stages of succession, turbid conditions resulting from the lack of substrate-stabilizing vegetation and increased phytoplankton growth appear to have discouraged C. vulgaris growth early in the season.     

Analyses of Phaseolus vulgaris L. and P. coccineus Lam. hybrids by RFLP: preferential transmission of P. vulgaris alleles

Summary Restriction fragment length polymorphism (RFLP) was determined among P. vulgaris genotypes and Phaseolus species using 19 probes. The incidence of polymorphism was high (70–86%) between species, but relatively low (22–26%) between genotypes of P. vulgaris. Suitable probes were identified for the analysis of P. vulgaris and P. coccineus hybrids. The segregation pattern in F₂ populations was Mendelian for two probes (LHB and VEE20) and non-Mendelian for GS-g, CHS, and CHI. Statistical analyses indicated gametic selection with preferential transmission of the P. vulgaris alleles, which may account for the selective recovery of P. vulgaris progeny types observed earlier. The available hybrids of P. vulgaris and P. coccineus and the high degree of interspecific RFLP will facilitate the construction of a linkage map for Phaseolus.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Farm pond restoration using Chara vulgaris vegetation

Four aged Madison County, New York farm ponds were selected to see if various treatments could be used to restore the water quality. One pond was untreated and used as a control; another pond was partially drained and exposed to the drying and oxidizing effects of the air over the fall and winter; the other two ponds were drained and the accumulated sediment removed by bulldozing. In these latter two ponds, Chara vulgaris vegetation was inoculated following the restoration process. C. vulgaris growth rapidly became the dominant producer where this inoculation was accomplished in the fall of 1976, and it is expected that the other pond will also become a C. vulgaris pond in 1978 — after its oogonia have undergone the requisite winter dormancy period.Early C. vulgaris growth was found to be associated with clear water conditions and lessened phytoplankton growth; short, bushy, light-inhibited growth by the algae stabilized the bottom against wind-caused turbidity because of its rhizoidal growth within the substrate. Pioneer C. vulgaris growth was also found to be highly productive, significantly lowering the pond's CO₂ readings.Investigators of aquatic systems are cautioned to be cognizant of the effect of epiphytic growth on successional events in such environments. Such epiphytes are surely important, if not prime, causes of the demise of various aquatic macrophytes.The partial draining and exposing of a pond over the fall and winter did not yield significantly improved water conditions.     

New Techniques for the Estimation of Naturally Occurring Brassinosteroids

We have developed enzyme-linked immunosorbent assays (ELISAs) for measuring 24-epicastasterone and related brassinolide analogs, with detection ranges of 0.005 to 50 pmoles. Polyclonal antibodies used in these assays were raised against 24-epicastasterone carboxymethyloxime-bovine serum albumin conjugates and were found to have high specificity for 24-epibrassinosteroids. Natural brassinosteroids (BRs), such as brassinolide and 24-epibrassinolide, exhibited relatively high cross-reactivities with the generated antibodies, whereas other BR analogs with β-oriented hydroxyl groups at C-2, C-3, C-22, and C23 lacked immunoreactivity. Through the use of internal standardization, dilution assays, recovery of authentic [³H]24-epicastasterone, and immunohistograms, the ELISAs have been shown to be applicable for estimating 24-epibrassinosteroid levels in crude plant extracts. To analyze brassinosteroids in tissues from young bean (Phaseolus vulgaris L., cv. Pinto), Daucus carota ssp.sativus plants and Arabidopsis thaliana L. Heynh. seedlings, and rape (Brassica napus L.) pollen, the extracts were fractionated by high performance liquid chromatography (HPLC) and the resulting fractions were analyzed by the ELISA method. Immunohistogram ELISA analysis of HPLC fractions indicated that major peaks of immunoreactivity co-chromatographed with the labeled and unlabeled 24-epibrassinolide. A highly sensitive electrospray ionization mass spectrometry (MS) technique (LOD: 50 fmol) was also developed and the results obtained by the HPLC-ELISA and HPLC-MS approaches were compared.     

Estimation of the Gelatinization Temperature of Noodles from Water Sorption Curves under Temperature-Programmed Heating Conditions

Both free and conjugated brassinosteroids (BRs) in the pollen and anthers of Erythronium japonicum Decne. were investigated. As a free form of BRs, typhasterol was identified by GC-MS. Polar conjugated BRs occurred only in the anther, and non-polar con-jugated BRs occurred both in the pollen and mainly in the anther.

BR parts of acid hydrolysis of the former were identified to be teasterone (major) and castasterone (minor). Those of alkaline hydrolysis of the latter were identified to be typhasterol (major) and teasterone (minor).     

Radioimmunoassay for brassinosteroids and its use for comparative analysis of brassinosteroids in stems and seeds ofPhaseolus vulgaris

Antiserum against the brassinosteroid (BR), castasterone, was produced by immunizing a rabbit with castasterone-carboxymethoxylamine oxime conjugated with bovine serum albumin (BSA). In a radioimmunoassay (RIA), the antiserum recognized a range of naturally occurring BRs with varying specificities. Detection limits of castasterone and brassinolide were approximately 0.3 pmol. This RIA system was successfully used for analyzing endogenous BRs in seeds and stems ofPhaseolus vulgaris L., and showed that stems are quite different from seeds in terms of the species and quantity of the endogenous BRs.     

Restriction fragment length polymorphisms of the phytohemagglutinin genes inPhaseolus andVigna (Leguminosae)

Restriction fragment length polymorphisms at the phytohemagglutinin (PHA) locus were determined among 21 genotypes ofPhaseolus vulgaris, P. coccineus, P. acutifolius, P. lunatus, and threeVigna species, using five restriction enzymes and one double digestion, in order to provide molecular evidence for their genetic relatedness. The dissimilarity between genotypes was estimated from binary RFLP data. The dissimilarity was high among species (from 0.75 to 0.95), and of variable extent among genotypes of the same species (0.33–0.89). InP. vulgaris, two different DNA hybridization patterns were found, giving further evidence for two major gene pools in that species. The restriction patterns ofP. vulgaris var.aborigineus, the putative ancestral form ofP. vulgaris, exhibit clear homology toP. vulgaris genotypes. An undefined landrace from Taiwan could be identified as aP. vulgaris genotype. RFLP-based trees for the phytohemagglutinin genes of the species studied were computed with several distance matrix and parsimony methods.     

DNA restriction fragment length polymorphisms correlate with isozyme diversity in Phaseolus vulgaris L.

Summary Genetic variation in Phaseolus vulgaris L. (P. vulgaris) was investigated at the isozyme and DNA levels. We constructed a library of size-selected Pst I clones of P. vulgaris nuclear DNA. Clones from this library were used to examine 14 P. vulgaris accessions for restriction fragment length polymorphisms (RFLPs). DNAs from each accession were analyzed with three restriction enzymes and 18 single copy probes. The same accessions were also examined for variability at 16 isozyme loci. Accessions included four representatives of the T phaseolin group and five representatives each of the C and S phaseolin groups. One member of the S group (the breeding line XR-235-1-1) was derived from a cross between P. vulgaris and P. coccineus. Isozymes and RFLPs revealed very similar patterns of genetic variation. Little variation was observed among accessions with C and T phaseolin types or among those with the S phaseolin type. However, both isozyme and RFLP data grouped accessions with S phaseolin separately from those accessions with C or T phaseolin. The highest degree of polymorphism was observed between XR-235-1-1 and members of the C/T group. RFLP markers will supplement isozymes, increasing the number of polymorphic loci that can be analyzed in breeding, genetic, and evolutionary studies of Phaseolus.     

Chloroplast DNA and isozyme evidence on the evolution ofSenecio vulgaris (Asteraceae)

Chloroplast DNA (cpDNA) and isozyme variation were analyzed over a range of populations of two infraspecific taxa of the tetraploidSenecio vulgaris. The isozyme data were supportive of the hypothesis that the weedy and cosmopolitanS. vulgaris var.vulgaris is an evolutionary derivative ofS. vulgaris subsp.denticulatus from the coasts of W Europe and montane altitudes in S Spain and Sicily. The two taxa exhibited a very high genetic identity with subsp.denticulatus containing slightly more isozyme diversity than was found in var.vulgaris. — Three cpDNA haplotypes (A, B, C) already known from other Mediterranean diploid species ofSenecio were resolved in var.vulgaris, and an additional fourth haplotype (E) was found in subsp.denticulatus. Two alternative hypotheses were chosen to account for the origin and maintenance of the observed cpDNA composition ofS. vulgaris. It either reflects (1) the retention of an ancestral polymorphism which stems from the recurrent and polytopic formation of ancestral tetraploid lineages; or (2)S. vulgaris originally was characterized by haplotype E, and haplotypes A, B and C were acquired through repeated introgressive hybridization with related diploid species. The finding that very low levels of nuclear (isozyme) diversity were present in both taxa ofS. vulgaris examined supports the second of these two hypotheses; however, more detailed analysis of nuclear genetic diversity is required before a firm conclusion can be reached on this matter.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Metabolic fingerprinting for bio-indication of nitrogen responses in <Emphasis Type="Italic">Calluna vulgaris</Emphasis> heath communities

Increased atmospheric deposition of nitrogen (N) over the last 50 years is known to have led to deleterious effects on the health of Calluna vulgaris heathland, with increased proliferation of grasses and loss of species diversity. However, currently it is difficult to attribute damage specifically to N deposition rather than other drivers of change such as inappropriate management. Metabolic fingerprinting using FT-IR offers a rapid, cost-effective and “holistic” means for quantifying foliar biochemistry responses specifically to N deposition. To test the potential of this approach we used a long term lowland heath N addition study in Chesire, England. FT-IR spectra of treated C. vulgaris shoot material showed that responses were detectable above 20 kg N ha⁻¹ year⁻¹. Differentiation was also evident in C. vulgaris metabolic fingerprints due to additional watering. We have shown that FT-IR is able to identify biochemical variations in C. vulgaris related to increases in received N and water. This technique therefore provides a sensitive measure of biochemical change in response to N addition, and allows development towards predictive modelling of N deposition at the landscape level.     

Plant species effect on the spatial patterns of soil properties in the Mu-us desert ecosystem,Inner Mongolia,China

Although Artemisia ordosica Krasch. and Sabina vulgaris Ant. are the dominant shrub species in the Mu-us desert ecosystem, they differ in their botanical traits. We investigated the spatial patterns of soil properties using geostatistical analysis to examine the effect of plant species on these spatial patterns. Comparison among three microsite types (under A. ordosica, under S. vulgaris, and the opening between vegetation) showed that A. ordosica generally had less effect than S. vulgaris on local soil properties. The long life-span, prostrate life-form, and evergreen leaf-habit of S. vulgaris may lead to a higher accumulation of organic and fine materials under S. vulgaris. The range of spatial autocorrelation found in the mass of organic matter on the soil surface was smaller than that of the coverage of S. vulgaris (11.5 m) which corresponded to the canopy patch size of this species, and was longer than the canopy patch size of A. ordosica. The ranges of total C and N, and pH (11.7–15.6 m) were similar to that of S. vulgaris. The range of available P (106.3 m) was comparable to that of the coverage of A. ordosica (86.2 m) considered to be the scale of the distribution of this species. The ranges of silt+clay and exchangeable K, Ca, and Mg (31.0–46.7 m) were not related to plant presence, and were similar to that of topography (43.1 m).     

Identification and expression studies of a catalase and a bifunctional catalase-peroxidase in Frankia strain R43

This study investigated the uptake of ¹³⁴Cs by Lactuca sativaL., Silybum marianumGaertn., Centaurea cyanusL., Carthamus tinctorius L. from the Asteraceae, and Beta vulgaris L. var. `Lutiancai' and Beta vulgaris L. var. `Hongtiancai' from the Chenopodiaceae grown in two widely-distributed soils (a paddy soil and a red soil) in South China. The results showed that the plants growing on the paddy soil had a relatively high yield and low [¹³⁴Cs] while those growing on the red soil showed the opposite trend. The accumulation of ¹³⁴Cs was dependent on plant species and soil types. For the paddy soil, mean values for [¹³⁴Cs] were higher for the species of the Asteraceae (ranging from 165 to 185 Bq g^–1) than for those of the Chenopodiaceae (less than 140 Bq g^–1). For the red soil, S. marianumand C. cyanus of the Asteraceae had high average concentrations of ¹³⁴Cs ranging from 340 to 400 Bq g^–1 but L. sativa and C. tinctorius from the same family had low concentrations of ¹³⁴Cs ranging from 115 to 200 Bq g^–1 on a dry weight basis. B. vulgaris L. var. `Lutiancai' and Beta vulgaris L. var. `Hongtiancai' accumulated from 120 to 231 Bq ¹³⁴Cs g^–1 of plant shoot. The transfer factor values of ¹³⁴Cs for the studied species were in general higher in red soil than in paddy soil except C. tinctorius. All plant species from the Asteraceae family growing on the paddy soil had higher transfer factors than the B. vulgaris species. S. marianum, and C. cyanus growing on the red soil had TFs >1, being much higher than the B. vulgaris species. The results therefore showed that the plant species from the Asteraceae could accumulate higher concentrations of radiocesium than the Beta vulgaristhat has previously been suggested as a candidate for phytoremediation of radiocesium contaminated soils.     

Effects of phenolic compounds on growth and metabolic activities of Chlorella vulgaris and Scenedesmus bijugatus isolated from soil

Phenol and three nitrophenols (o-nitrophenol, m-nitrophenol, p-nitrophenol), commonly occurring pollutants in natural eco-systems, were tested for their toxic effects on soil isolates of Chlorella vulgaris and Scenedesmus bijugatus, growing under phototrophic, photoheterotrophic and heterotrophic conditions. The toxicity criteria included cell number, chlorophyll, total protein and carbohydrate content, ¹⁴CO₂ uptake and in vivo nitrate reductase activity. Both C. vulgaris and S. bijugatus were sensitive to the pollutants when the cultures were grown under phototrophic or heterotrophic conditions. However, the toxicity was found reversed or alleviated upon photoheterotrophic growth of the cultures. Transmission electron microscope studies revealed various cytological abnormalities in C. vulgaris in the presence of phenolics at algistatic levels.     

Carbon dioxide fixation by Chlorella kessleri, C. vulgaris, Scenedesmus obliquus and Spirulina sp. cultivated in flasks and vertical tubular photobioreactors

CO₂ at different concentrations were added to cultures of the eukaryotic microalgae, Chlorella kessleri, C. vulgaris and Scenedesmus obliquus, and the prokaryotic cyanobacterium, Spirulina sp., growing in flasks and in a photobioreactor. In each case, the best kinetics and carbon fixation rate were with a vertical tubular photobioreactor. Overall, Spirulina sp. had the highest rates. Spirulina sp., Sc. obliquus and C. vulgaris could grow with up to 18% CO₂.