Molecular mechanisms underlying Grateloupia imbricata (Rhodophyta) carposporogenesis induced by methyl jasmonate

When applied in vitro, methyl jasmonate is sensed by the red seaweed Grateloupia imbricate, substantially and visually affecting its carposporogenesis. However, although there is some understanding of the morphological changes induced by methyl jasmonate in vitro, little is known about the genes that are involved in red seaweed carposporogenesis and how their protein products act. For the work reported herein, the expression of genes in red seaweed that encode enzymes involved in the synthesis of methyl jasmonate (jasmonic acid carboxyl methyl transferase and a putative methyl transferase) was monitored. Additionally the genes involved in oxidation (cytochrome P450 and WD40), jasmonate synthesis, signal transduction, and regulation of reactive oxygen species (MYB), and reproduction (ornithine decarboxylase) were monitored. To determine when or if the aforementioned genes were expressed during cystocarp development, fertilized and fertile thalli were exposed to methyl jasmonate and gene expression was measured after 24 and 48 h. The results showed that methyl jasmonate promoted differential gene expression in fertilized thalli by 24 h and upregulated expression of the ornithine decarboxylase gene only by 48 h in fertile thalli (0.75 ± 003 copies · μL^?1 at 24 h vs. 1.11 ± 0.04 copies · μL^?1 at 48 h). We conclude that Ornithine decarboxylase expression involves methyl jasmonate signaling as well as development and maturation of cystocarps.     

Occurrence of jasmonates during cystocarp development in the red alga Grateloupia imbricata

In this study, we highlight the effects of methyl jasmonate (MeJa) on cystocarp development in the red macroscopic alga Grateloupia imbricata. In G. imbricata, jasmonate release is related to the reproductive state, as fertile thalli (i.e., those that have cystocarps) released significant amounts of this volatile compound (1.27 ± 0.20 mM · mg fw^?1 · h^?1) compared with infertile thalli (0.95 ± 0.12 mM · mg fw^?1 · h^?1). Treating G. imbricata thalli with MeJa revealed a significant increase in cystocarp number (1.5 ± 0.27 cystocarps · mm^?2), which was ~7.5‐fold greater than in untreated thalli (0.2 ± 0.07 cystocarps · mm^?2). Maturation was completed within 48 h with MeJa treatment, a shortening of the typical >3‐week maturation period, and included the opening of cystocarps and the presence of dehiscent cavities. Release rates of jasmonates after exogenous MeJa treatment were also modified based on the cystocarp maturation level. All of these effects were reduced in the presence of phenidone, which blocks MeJa production, indicating that the MeJa action is genuine. The effects of MeJa during cystocarp maturation were not replicated by derivatives of reactive oxygen species from the same jasmonic acid biosynthetic pathway, as the activities of scavenger enzymes and lipid peroxidation were unchanged between infertile and fertile thalli. Therefore, a reactive oxygen species–based mechanism is not involved during cystocarp development. We conclude that MeJa has an independent function as a growth regulator during G. imbricata reproduction.     

The polyamine spermine induces cystocarp development in the seaweed <Emphasis Type="Italic">Grateloupia</Emphasis> (Rhodophyta)

Polyamines such as putrescine, spermidine and spermine are ubiquitous aliphatic amines involved in reproductive events in plants and algae, and first become evident through changes in endogenous levels during reproductive development. To examine whether the differences observed in polyamines, during carposporogenesis, in the red alga Grateloupia, followed a specific pattern as is seen in other organisms, infertile axes (i.e. not showing cystocarps) were excised from the same holdfast of female fertilized individuals (i.e. showing cystocarps in other axes), and cultivated until the cystocarps became visible. Changes in the endogenous levels of free putrescine, spermidine and spermine were monitored over the 8 days of culture. The activity of enzymes related to polyamine metabolism, such as l-ornithine decarboxylase (ODC), diamine oxidase and polyamine oxidase, was measured at the beginning and end of the experimental period. Up to 50% of the infertile axes became fertile and produced cystocarps at a density of 1.91 ± 0.1 cystocarps mm⁻² after 8 days. The endogenous content of spermine increased markedly over the first 5 days of culture, then decreased to the initial level by day 8. Spermidine followed a similar pattern to spermine, whereas putrescine remained at high levels, until day 5 when it decreased abruptly. The activity of ODC was less on day 8 than on day 0, whereas the activities of diamine oxidase and polyamine oxidase increased. In parallel experiments with explants from infertile axes, exogenously added spermine (10⁻⁶ M) increased the number of cystocarps, and reversed the effect of cyclohexylamine (CHA), which is known to inhibit polyamine synthesis in Grateloupia. Serial sectioning and microscopic observation of specimens from explants cultivated in 10⁻⁶ M spermine indicated that cystocarp development was induced. The results suggest that, during transition from infertile to fertile spermine is accumulated, thus favouring the development of cystocarps, given the presumed role of spermine as an inducing agent.     

POLYAMINES INFLUENCE MATURATION IN REPRODUCTIVE STRUCTURES OF GRACILARIA CORNEA (GRACILARIALES,RHODOPHYTA)1

Naturally occurring levels of putrescine, spermidine, and spermine were analyzed in female gametophyte (nonfertilized) and reproductive tissues (cystocarps) at two different stages of development in the marine red algae Gracilaria cornea J. Agardh. Endogenous polyamine levels changed at differential stages of cystocarp maturation. Highest polyamine values were found on tissue from the early post‐fertilization stage, decreasing as the cystocarp matured. Incubation experiments revealed that exogenous polyamines induced cystocarp maturation and promoted carpospore liberation, developing cell masses within 4 to 7 days in treatments with spermine. This is the first report on the effect of polyamines on cystocarp maturation in marine algae.     

Effect of methylglyoxal bis-(guanylhydrazone) on polyamine and ethylene biosynthesis of apple fruit after harvest

Polyamine and ethylene both play important roles in fruit ripening, whose biosynthetic pathways share a common substrate, S-adenosylmethionine (SAM). To unravel the interrelationship between polyamine and ethylene, their metabolism and expression of relevant genes were investigated in apple fruit (Malus domestica Borkh.) treated with methylglyoxal bis-(guanylhydrazone) (MGBG). The MGBG-treated fruit had higher ethylene production until 16 days after treatment (DAT) with preceding accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) than control fruit and then decreased to nearly the same level as control. Ethylene promotion at the early stage by MGBG was accompanied by increased expression of apple ACC synthase (Md-ACS1) and ACC oxidase (MdACO). The expression of apple SAM synthase (MdSAMS) in MGBG-treated fruit was slightly higher than that in control. On the other hand, significant changes in free polyamine titers were observed at some stages, but the changes did not show consistent trends. Based on these observations, possible relationship between polyamine and ethylene pathways was discussed.     

Non-random mating in controlled multiple-donor crosses in Gracilaria gracilis (Gracilariaceae,Rhodophyta)

Crosses were performed to identify the sources of variation in zygote production (via cystocarp production) in Gracilaria gracilis, a red haploid-diploid seaweed. First, because male gametes are short-lived (<6?h), the rate of gamete encounters was evaluated in a time-course experiment. Second, the effect of water motion on gamete encounters was assessed by introducing turbulent eddies in the crossing tank and by comparing fertilization rates with and without this added turbulence. Third, variation due to individual performance was explored by performing multiple-donor crosses using 12 males and 12 females from three populations. Paternity of cystocarps produced in these crosses was determined using microsatellite markers. The results show that cystocarp yield increased with exposure time: fertilization occurred in as little as 15?min after the introduction of male branches into the crossing tank and maximum cystocarp production values were observed at 6?h. There were no significant differences in cystocarp production between the two turbulence levels. On the other hand, cystocarp production was highly influenced by male and female parental identities and to a lesser degree by an interaction between the male and female parents. The variation in cystocarp production according to male and female identity was not due to population origin as there was no difference between intra- and inter-population crosses. Thus nonrandom mating occurs in controlled conditions and arises from differential performance in G. gracilis. There was a strong deviation from equality of male performance, implicating post-adhesion events and/or male gamete production as important in generating non-random mating. Consequently, non-random mating may play a role in the evolution of mating patterns in G. gracilis.     

Intrageneric differences in cystocarp structure in Gelidium and Pterocladia

Unilocular cystocarps, with ostioles opening to one frond surface, have traditionally distinguished Pterocladia from Gelidium, described as having bilocular cystocarps, with ostioles opening to both surfaces; however, unequally developed locules have been described in Pterocladia and differences in cystocarpic architecture between Pterocladia capillacea and the type species of the genus, P. lucida, have been recently found. As heterogeneity in cystocarp architecture raises questions of basic intergeneric distinction, a survey of reproductive morphology of species in both genera is presented in this study. Six morphologically-different types of cystocarps are distinguished among the five species of Pterocladia and the seven species of Gelidium examined.     

EFFECTS OF ETHYLENE ON TETRASPOROGENESIS IN PTEROCLADIELLA CAPILLACEA (RHODOPHYTA)1

The effects of ethylene (C₂H₄) on tetrasporogenesis of the red seaweed Pterocladiella capillacea (S. G. Gmelin) Bornet were investigated. Ethylene is a gaseous hormone that is involved in a variety of physiological processes (e.g., flowering, fruit abscission) in higher plants. To study the effects of ethylene on the reproduction of the red seaweed P. capillacea, immature tetrasporophytic thalli were exposed to a flow of ethylene for different time periods. Maximum maturation of tetrasporangia was observed at 7 d in thalli exposed to ethylene for 15 min. This maturation was accompanied by a significant increase in the free fraction of putrescine (Put) and a 5‐fold increase in the level of total RNA. These changes were specifically due to ethylene since they were blocked by the presence of the ethylene perception inhibitor silver thiosulphate (STS). Moreover, P. capillacea was determined to produce ethylene at a rate of 1.12 ± 0.06 nmol ethylene · h^?1· g^?1 fresh weight (fwt) with specific activities for 1‐aminocyclopropane‐1‐acrylic acid (ACC) synthase of 11.21 ± 1.19 nmol ethylene · h^?1· mg^?1 protein and for ACC oxidase (ACO) of 7.12 ± 0.11 nmol ethylene · h^?1· mg^?1 protein. We conclude that ethylene may indeed be a physiological regulator of tetrasporogenesis in this red seaweed.     

CARRAGEENANS BIOSYNTHESIZED BY CARPOSPOROPHYTES OF RED SEAWEEDS GIGARTINA SKOTTSBERGII (GIGARTINACEAE) AND GYMNOGONGRUS TORULOSUS (PHYLLOPHORACEAE)1

Carrageenans biosynthesized by gametophytic and tetrasporic plants of seaweeds belonging to the Gigartinaceae and Phyllophoraceae are different: gametophytes produce carrageenans of the kappa family, whereas lambda‐carrageenans are extracted from tetrasporophytes. For Gigartina skottsbergii Setchell and Gardner and Gymnogongrus torulosus Hooker et Harvey, mature cystocarps were isolated and carrageenans were extracted. Structural determination by methylation analysis, Fourier transform infrared spectroscopy, and ¹³C‐NMR spectroscopy showed that they were kappa/iota‐carrageenans. For the extract obtained from cystocarps of Gigartina skottsbergii with water at room temperature, the ratio kappa:iota was 1:0.30 and at 90° C was 1:0.43; significant amounts of precursors were also present. The extract obtained from cystocarps of Gymnogongrus torulosus at 90° C showed prevalence of iota‐carrageenans (ratio kappa:iota 1:1.21). These extracts are similar to the polysaccharides produced by gametophytes of these seaweeds. For Gigartina skottsbergii, it was possible to separate the pericarpic tissue from the carposporophyte. Thus, they were extracted separately, and the carrageenans isolated were studied as described before, obtaining similar conclusions. These results clearly show that whereas the carposporophytes are located inside the cystocarp, they produce carrageenans of the kappa family despite of being diploid cells.     

Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> infection

Background  

Plant cytochrome P450 monooxygenases (CYP) mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to Xylella fastidiosa (Xf) infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B gene in both disease resistant and susceptible grapevines.     

Acclimation to UV irradiance in Gracilaria chilensis Bird,McLachlan & Oliveira (Gigartinales,Rhodophyta)

Photoautotrophs can cope with an increase in ultraviolet (UV) irradiance in the aquatic environment, through protection and acclimation mechanisms (i.e. synthesis of UV-absorbing compounds). This capacity has been proven to vary according to the organism's sensitivity. To quantify variations of this capacity between the different parts of macroalgae, an in vitro study was performed with the tips, cystocarps and thalli segments of Gracilaria chilensis. Whole algae incubated during 3 days at high and low PAR, supplying UV B (4.6 µW cm^–2) during 2 hours showed, as predicted, an increase in absorption (OD) at 320 nm of the different parts, after the first day of exposure to UV B. The tips presented the highest increase in the standardized OD at 320 nm relative to cystocarps and thalli segments; their mean percentage of increase was 38% and 29% at low and high PAR, respectively. The lowest sensitivity was consistently found in the thalli segments, while the highest was in the tips. The tips are important for growth and therefore they play a major role in the maintenance of the Gracilaria populations. Acclimation mechanisms that occurred in a short time scale, and mainly in the tips, may allow Gracilaria to have an almost immediate protection to increases in UV B fluxes.     

Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a β-carotene 3-hydroxylase/4-ketolase

A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a β-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the β-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this β-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from β-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of β-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both β-ionone ends.     

Freezing tolerance of <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Bangiales,Rhodophyta) gametophyte assessed by chlorophyll fluorescence

In January and February 2010, heavy sea ice formed along the coast of the Bohai Sea and the northern Yellow Sea, China. Intertidal organisms were subjected to serious freezing stress. In this study, we investigated the freezing tolerance of the upper intertidal economic seaweed Porphyra yezoensis. The maximum photochemical efficiency of PS II (F _v/F _m) in undehydrated thalli remained high after 24 h at −2°C and that in dehydrated thalli decreased in a proportion to thallial water loss. F _v/F _m dropped sharply after 24 h at −20°C, regardless of absolute cellular water content (AWC). The F _v/F _m in frozen thalli recovered rapidly at 0–20°C. A wide range of water loss in the thalli enhanced their tolerance to freezing. F _v/F _m values in undehydrated thalli dropped sharply after 3 d at −2°C or 10 d at −20°C while those in dehydrated thalli (20–53% AWCs) remained at high levels after 9 d at −2°C or 30 d at −20°C. These results indicate that P. yezoensis has high freezing tolerance by means of dehydration during the ebb tide and rapid recovery of F _v/F _m from freezing. A strategy of P. yezoensis industry to avoid heavy loss during freezing season is discussed based on these findings.     

Isolation and characterization of RDX-degrading <Emphasis Type="Italic">Rhodococcus</Emphasis> species from a contaminated aquifer

Groundwater contamination by the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a global problem. Israel’s coastal aquifer was contaminated with RDX. This aquifer is mostly aerobic and we therefore sought aerobic bacteria that might be involved in natural attenuation of the compound in the aquifer. RDX-degrading bacteria were captured by passively sampling the indigenous bacteria onto sterile sediments placed within sampling boreholes. Aerobic RDX biodegradation potential was detected in the sediments sampled from different locations along the plume. RDX degradation with the native sampled consortium was accompanied by 4-nitro-2,4-diazabutanal formation. Two bacterial strains of the genus Rhodococcus were isolated from the sediments and identified as aerobic RDX degraders. The xplA gene encoding the cytochrome P450 enzyme was partially (~500 bp) sequenced from both isolates. The obtained DNA sequences had 99% identity with corresponding gene fragments of previously isolated RDX-degrading Rhodococcus strains. RDX degradation by both strains was prevented by 200 μM of the cytochrome P450 inhibitor metyrapone, suggesting that cytochrome P450 indeed mediates the initial step in RDX degradation. RDX biodegradation activity by the T7 isolate was inhibited in the presence of nitrate or ammonium concentrations above 1.6 and 5.5 mM, respectively (100 mg l⁻¹) while the T9N isolate’s activity was retarded only by ammonium concentrations above 5.5 mM. This study shows that bacteria from the genus Rhodococcus, potentially degrade RDX in the saturated zone as well, following the same aerobic degradation pathway defined for other Rhodococcus species. RDX-degrading activity by the Rhodococcus species isolate T9N may have important implications for the bioremediation of nitrate-rich RDX-contaminated aquifers.     

Floating period of Sargassacean thalli estimated by the change in density

The density (fresh weight/volume) of the detached thalli of four sargassacean species (Myagropsis myagroides, Sargassum horneri, S. patens, S. siliquastrum) was measured weekly to determine their floating period. Thalli of the four species were collected with respect to the difference in their reproductive stage, and their floating abilities at different stages were determined. Floating thallus was ballasted with pre-weighed leads until the final addition of a weight caused sinking. The density and floating period of sargassacean thalli were dependent on the species and the specimens’ reproductive stage. The floating period of thalli detached before maturation was estimated to be between 4 and 14 weeks. However, thalli detached during maturation had a floating period of between 1 and 8 weeks, and those detached after maturation sank within 2 weeks. Sargassum horneri had the lowest density and the longest floating period among the four species investigated and this may explain the high abundance of S. horneri in floating seaweeds observed in the Sea of Japan.     

Gibberellin Biosynthetic Pathway inGibberella fujikuroi:Evidence for a Gene Cluster

Differential screening of aGibberella fujikuroicDNA library was used to successfully clone and identify genes involved in the pathway of gibberellin biosynthesis. Several cDNA clones that hybridized preferentially to a cDNA probe prepared from mycelium induced for gibberellin production were isolated and characterized. The deduced amino acid sequences of two (identical) clones contained the conserved heme-binding motif of cytochrome P450 monooxygenases (FXXGXXXCXG). One of these cDNA fragments was used as a homologous probe for the screening of a genomic library. A hybridizing 6.7-kb genomicSalI fragment was cloned into pUC19. The sequencing of this clone revealed that a second cytochrome P450 monooxygenase gene was closely linked to the first one. Since at least four cytochrome P450 monooxygenase-catalyzed steps are involved in the synthesis of gibberellins, chromosome walking was performed to find a further gene of this family or other genes involved in gibberellin pathway. Next to the two P450 monooxygenase genes, a putative geranylgeranyl diphosphate synthase gene, the copalyl diphosphate synthase gene, which is the first specific gene of the gibberellin pathway, and a third P450 monooxygenase gene were identified. These results suggest that at least some of the genes involved in the biosynthesis of gibberellins are closely linked in a gene cluster inG. fujikuroi,as has been recently found for other “dispensable” pathways in fungi.     

Alkane-induced expression,substrate binding profile,and immunolocalization of a cytochrome P450 encoded on the <Emphasis Type="Italic">nifD</Emphasis> excision element of <Emphasis Type="Italic">Anabaena</Emphasis> 7120

Background  

Alkanes have been hypothesized to act as universal inducers of bacterial cytochrome P450 gene expression. We tested this hypothesis on an unusual P450 gene (cyp110) found on a conserved 11 kilobase episomal DNA element of unknown function found in filamentous cyanobacteria. We also monitored the binding of potential substrates to the P450 protein and explored the distribution of P450 protein in vegetative cells and nitrogen-fixing heterocysts using immuno-electron microscopy.