Biocatalytic Regioselective O-acylation of Sesquiterpene Lactones from Chicory: A Pathway to Novel Ester Derivatives.

We report the first biocatalytic modification of sesquiterpene lactones (STLs) found in the chicory plants, specifically lactucin (Lc), 11ß,13-dihydrolactucin (DHLc), lactucopicrin (Lp), and 11ß,13-dihydrolactucopicrin (DHLp). The selective O-acylation of their primary alcohol group was carried out by the lipase B from Candida antarctica (CAL-B) using various aliphatic vinyl esters as acyl donors. Perillyl alcohol, a simpler monoterpenoid, served as a model to set up the desired O-acetylation reaction by comparing the use of acetic acid and vinyl acetate as acyl donors. Similar conditions were then applied to DHLc, where five novel ester chains were selectively introduced onto the primary alcohol group, with conversions going from >99 % (acetate and propionate) to 69 % (octanoate). The synthesis of the corresponding O-acetyl esters of Lc, Lp, and DHLp was also successfully achieved with near-quantitative conversion. Molecular docking simulations were then performed to elucidate the preferred enzyme-substrate binding modes in the acylation reactions with STLs, as well as to understand their interactions with crucial amino acid residues at the active site. Our methodology enables the selective O-acylation of the primary alcohol group in four different STLs, offering possibilities for synthesizing novel derivatives with significant potential applications in pharmaceuticals or as biocontrol agents.

Sexual reproduction of Zymoseptoria tritici on durum wheat in Tunisia revealed by presence of airborne inoculum, fruiting bodies and high levels of genetic diversity.

Septoria tritici blotch (STB) caused by the heterothallic ascomycete Zymoseptoria tritici is currently one of the most devastating diseases of wheat worldwide. The extent of sexual reproduction of this pathogen is well documented on bread wheat, but not on durum wheat. The objective of the present study was to quantify the occurrence of Z. tritici sexual reproduction on durum wheat in the Tunisian environment. The assessment was undertaken using a triple approach combining fruiting body assessment, ascospore trapping and population genetic analyses. The results highlighted the formation of pseudothecia on leaves and stubble from the autumn until the end of the growing season. Likewise, qPCR monitoring highlighted a constant release of Z. tritici airborne inoculum during the wheat-growing season, with a peak of production at the end of the season. Genetic investigations using microsatellites revealed high levels of gene and genotypic diversities, an equal distribution of mating types, and a lack of genetic clustering within and between growing seasons. Taken together, these findings indicate that Z. tritici undergoes sexual reproduction on durum wheat in Tunisia at least to the same extent than on bread wheat in Western Europe, and that the dry and warm climate does not affect the mating process of the fungus. Frequent occurrence of sexual reproduction is a valuable knowledge to take into account in STB control strategies on durum wheat.

A glutathione S-transferase cDNA identified by mRNA differential display is upregulated during somatic embryogenesis in Cichorium.

CHI-GST1, a cDNA encoding a glutathione S-transferase, was isolated by differential display in leaf tissues of chicory, during the early stages of somatic embryogenesis. Expression analysis of the gene by Northern blot indicated that the transcript accumulation is specific of the leaf developing somatic embryogenesis and is not observed in leaf tissue of the non-embryogenic cultivar.

Arabinogalactan-proteins in Cichorium somatic embryogenesis: effect of beta-glucosyl Yariv reagent and epitope localisation during embryo development.

Direct somatic embryogenesis was induced in root tissues of the Cichorium hybrid '474' (C. intybus L. var. sativum x C. endivia L. var. latifolia). Addition of beta-D-glucosyl Yariv reagent (betaGlcY), a synthetic phenylglycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in a concentration-dependent manner with complete inhibition of induction occurring at 250 microM betaGlcY. The AGP-unreactive alpha-D-galactosyl Yariv reagent had no biological activity in this system. Upon transfer of 250 microM betaGlcY-treated roots to control conditions, somatic embryogenesis was recovered with a time course similar to that of control roots. The betaGlcY penetrated roots and bound abundantly to developing somatic embryos, to the root epidermis and the stele. Immunofluorescence and immunogold labelling using monoclonal antibodies (JIM13, JIM16 and LM2) revealed that AGPs were localised in the outer cell walls peripheral cells of the globular embryo. A spatio-temporal expression of AGPs appeared to be associated with differentiation events in the somatic embryo during the transition from the globular stage to the torpedo stage. To verify betaGlcY specificity, molecules that bound betaGlcY were extracted from treated conditioned medium and identified as AGPs by using the same monoclonal antibodies. In addition, AGPs were found to be abundantly present in the medium during embryogenic culture. All of these results establish the implication of AGPs in embryo development, and their putative role in somatic embryogenesis is discussed.

Three major somatic embryogenesis related proteins in Cichorium identified as PR proteins.

In Cichorium hybrid clone '474' (C. intybus L., var. sativum x C. endivia L., var. latifolia), the direct somatic embryogenesis process in leaf tissues is accompanied by an overall increase in the amount of proteins secreted into the culture medium. Amongst these, three major protein bands of 38 kDa, 32 kDa and 25 kDa were found in the conditioned media. These extracellular protein bands accumulated in the medium of the embryogenic Cichorium hybrid up to 8-fold compared with those in the medium of a nonembryogenic variety. 32 and 25 kDa proteins were purified from the medium and their identities were determined as already described for 38 kDa beta-1,3-glucanases. To investigate their possible function in somatic embryogenesis, peptide sequences, serological relationships or biochemical properties revealed that there were at least two acidic chitinases of 32 kDa and one glycosylated osmotin-like protein of 25 kDa in the embryogenic culture medium. Comparing the amounts of the 38 kDa glucanases, the 32 kDa chitinases, and the 25 kDa osmotin-like protein present in the conditioned media of the embryogenic '474' hybrid and of a non-embryogenic variety, a 2-8-fold higher accumulation of these proteins was observed in the embryogenic hybrid culture medium. This may suggest that part of the accumulation of these three pathogenesis-related (PR) proteins could be correlated with the somatic embryogenesis process. Their possible involvement in this developmental process is discussed.

Cloning of beta-1,3-glucanases expressed during Cichorium somatic embryogenesis.

Three different beta-1,3-glucanase cDNA fragments, CG1, CG2 and CG3, were obtained by RT-PCR from RNA isolated from Cichorium hybrid '474' leaf fragments cultured for 11 days under somatic embryogenesis-inducing conditions. When expressed in Escherichia coli the proteins encoded by the three cDNAs were recognized by antibodies raised against 38 kDa extracellular beta-1,3-glucanases studied previously (Helleboid et al., Planta 205 (1998) 56-63). The CG2 and CG3 cDNAs may represent expressed alleles of one gene because their sequences showed a very high identity (98.5%) and are only 70% identical with CG1. Southern blot analysis revealed the presence of 3-4 genes coding for beta-1,3-glucanases in the Cichorium genome. Expression analysis of the genes corresponding to the three clones analysed by semi-quantitative RT-PCR indicated that CG1 mRNAs were only detectable in Cichorium hybrid '474' leaf fragments from day 3 of somatic embryogenesis induction, whereas CG2-CG3 mRNAs were already present in non-induced leaf tissue of both the embryogenic hybrid '474' and a non-embryogenic genotype. The level of CG1 mRNAs was particularly high when embryogenic cells were dividing to produce embryos, and when the amount of callose deposited in cell walls surrounding embryogenic cells and young embryos decreased. These results indicate that expression of the CG1 gene is correlated to the somatic embryogenesis process and that it encodes a 38 kDa beta-1,3-glucanase protein that may be involved in the degradation of callose localized around embryogenic cells and young embryos. A full-length CG1 cDNA clone was obtained using 3' and 5' RACE-PCR, and its sequence revealed that it encodes a beta-1,3-glucanase that is equally homologous to both class III and class IV plant beta-1,3-glucanases.

A nonsymbiotic hemoglobin gene is expressed during somatic embryogenesis in Cichorium.

After differential screening of a cDNA library corresponding to genes expressed during the early stages of somatic embryogenesis in leaf tissue from the Cichorium hybrid '474' (C. intybus L., var. sativumxC. endivia L., var. latifolia) a nonsymbiotic hemoglobin cDNA was obtained. Studies of the expression of the gene corresponding to this clone by Northern blot analysis suggest that in Cichorium a nonsymbiotic hemoglobin gene is specifically expressed under somatic embryogenesis-inducing conditions, and that its expression is not related to stress caused by wounding or tissue culture conditions.

Extracellular beta-1,3-glucanases are induced during early somatic embryogenesis in Cichorium.

In leaf tissues of the Cichorium hybrid clone '474' (C. intybus L. var. sativum x C. endivia L. var. latifolia), the acquisition and expression of embryogenic competence was characterised by the appearance of 15 polypeptides (Boyer et al., 1993, Plant Sci 93: 41-53). The 38-kDa proteins were found to be abundantly present in conditioned embryogenic medium after the first division of the induced cells. These proteins seemed to be glycosylated as indicated by general carbohydrate detection methods. Internal amino-acid sequences obtained after microsequencing tryptic peptides appeared to be 36-57% homologous with plant beta-1,3-endoglucanases. In addition, these 38-kDa proteins were recognised by antibodies raised against the pathogenesis-related tobacco glucanase PR2a and their beta-1,3-glucanase activity was demonstrated by direct detection in polyacrylamide gels after electrophoresis. These results strongly suggested that the 38-kDa somatic-embryogenesis-related (SER) polypeptides are beta-1,3-glucanases. Moreover, the level of glucanase activity was nearly three times higher in the medium of the embryogenic '474' line than in the medium of a non-embryogenic line. The possible involvement of the extracellular 38-kDa proteins in callose degradation during somatic embryogenesis is discussed.

Stress proteins in the polychaete annelid Nereis diversicolor induced by heat shock or cadmium exposure.

We have exposed the polychaete annelid Nereis diversicolor to heat shock or cadmium. Two-dimensional electrophoresis and fluorography techniques indicated the synthesis by the worms of at least 15 stress proteins including the universal one referred to as 'stress 70' and a lot of low molecular weight (LMW) proteins. 'Stress 70', synthesized by Nereis diversicolor in response to both stressors, appeared on fluorograms as an array of three charge isomers. We observed that most of the LMW stress proteins built up in response to heat shock were different from those observed after cadmium exposure. Furthermore, Nereis, which resists high levels of cadmium, did not appear to synthesize metallothioneins, small proteins known to prevent cellular damage by sequestering toxic metal ions. As no cadmium-binding proteins were visualized on fluorograms, the mechanism by which Nereis resists cadmium injury remains to be disclosed.

Ectomycorrhizin Synthesis and Polypeptide Changes during the Early Stage of Eucalypt Mycorrhiza Development.

In functioning eucalypt ectomycorrhizas, biochemical alterations are accompanied by a differential accumulation of polypeptides including the synthesis of symbiosis-related proteins (JL Hilbert, Martin FM [1988] New Phytol 110: 339-346). In the present study, protein biosynthesis in the early stages of ectomycorrhiza formation on Eucalyptus globulus subsp. bicostata Kirkp. was examined using compatible and incompatible isolates of the basidiomycete Pisolithus tinctorius (Coker & Couch). Changes in polypeptide composition were observed within hours following contact of the compatible mycelium with the roots, well before the differentiation of typical symbiotic tissues. At this stage, at least seven symbiosis-related proteins (ectomycorrhizins) accumulated in root tissues. In vivo incorporation of [(35)S]methionine by ectomycorrhizas followed by electrophoresis of the labeled proteins revealed that most of these differences in polypeptide concentrations, including the ectomycorrhizin accumulation, are the result of differential protein biosynthesis rather than posttranslational modifications of the polypeptides. The initial development of eucalypt ectomycorrhizas, therefore, coincides with the synthesis of symbiosis-related proteins and the data presented here provide essential evidence to ascribe a functional developmental role to these proteins.

Estradiol secretion by granulosa cells from rats with four- or five-day estrous cycles: the development of responses to follicle-stimulating hormone versus luteinizing hormone.

The relative importance of LH vs. FSH in stimulating estradiol secretion by granulosa cells during follicular development was assessed in Sprague-Dawley rats with regular 4- or 5-day estrous cycles. At various times during diestrus (D-1200 h, D-2000 h) and proestrus (P-0800 h, P-1400 h, P-2000 h) granulosa cells were isolated from the presumptive preovulatory follicles. The cells were cultured with 0.5 microM testosterone and various doses of LH or FSH (0, 0.1, 1, 10, or 100 ng/ml). Media were collected and replaced daily for 3 days and measured for estradiol by RIA. Estradiol secretion in the absence of gonadotropins (endogenous aromatase activity) increased progressively with stage of the cycle. At earlier times of cell isolation (D-1200, D-2000, P-0800) secretion by cells from 5-day rats was greater relative to 4-day rats. Since estradiol production in the absence of gonadotropins declined progressively over each 3-day culture period, effects of the gonadotropins were most evident on the third day of culture when endogenous aromatase activity was low. On this day FSH consistently increased estradiol secretion above control levels. Sensitivity to FSH, as measured by the 50% maximally effective dose (ED50; approximately 1-3 ng FSH/ml), did not vary with cycle type or stage of the cycle and the response to FSH, in terms of 50% maximal estradiol secretion, was also relatively constant. In contrast, the effects of LH varied with cycle type and time of cell isolation. As follicles developed, cells from both 4- and 5-day rats became more sensitive to LH, as evidenced by a decline in the ED50 from approximately 32 ng LH/ml to approximately 3 ng/ml. This increase in sensitivity to LH is consistent with previous reports that the number of LH receptors on granulosa cells increases progressively during the final stages of follicular development. However, the increase in sensitivity to LH occurred earlier in cells from 5-day rats, relative to the expected next estrus (later, relative to the preceding estrus). Responsiveness to LH (50% maximal estradiol production) was relatively constant, except that it was lower in cells from 4-day rats isolated at D-1200. These results indicate that the LH receptors acquired by granulosa cells during diestrus and proestrus are functionally linked with aromatase activity and may, therefore, be important to the production of estradiol levels sufficient to elicit the LH surge.(ABSTRACT TRUNCATED AT 400 WORDS)

Progesterone secretion by granulosa cells from rats with four- or five-day estrous cycles: the development of responses to follicle-stimulating hormone, luteinizing hormone, and testosterone.

Changes in granulosa cell sensitivity and responsiveness to gonadotropins during the rat estrous cycle were studied by measuring progesterone (P) secretion in vitro in response to treatment with increasing doses of LH or FSH (0, 0.1, 1, 10, 100 ng/ml). The effect of testosterone [(T); 0.5 microM] on response to gonadotropins was also examined. Granulosa cells were isolated from the largest ovarian follicles of rats with 4- and 5-day estrous cycles at 0800 h, 1400 h, and 2000 h on proestrus and on the preceding day of diestrus at 1200 h and 2000 h. In rats with 5-day cycles, granulosa cells were also obtained at 1200 h on the first day of diestrus. Fifty percent maximal P production and 50% effective dose (ED50-dose of gonadotropin which elicited 50% maximal P production) were calculated from dose-response curves for LH and FSH and were used as measures of responsivity and sensitivity to gonadotropins, respectively. Basal P secretion and 50% maximal P secretion increased progressively as cells were isolated at later stages of follicular growth in both 4- and 5-day cycles. In cells from 5-day rats, however, basal and gonadotropin-stimulated P secretion were higher on the second day of diestrus than in cells from 4-day rats. By proestrus responsiveness was equal. Granulosa cell sensitivity to FSH was constant during 4- and 5-day cycles, as indicated by a lack of change in the ED50. Granulosa cell sensitivity to LH was lower than sensitivity to FSH on diestrus of both 4- and 5-day cycles. However, by the morning of proestrus sensitivity to LH increased and was similar to that for FSH. T increased basal P production only slightly, but synergized with both LH and FSH to stimulate 2-fold increases in 50% maximal P production by granulosa cells isolated at all times except 2000 h on proestrus, after the endogenous LH surge. T had no effect on the sensitivity (ED50) of granulosa cells to LH or FSH. In summary, granulosa cell responsiveness to LH and FSH increased in parallel during the final stages of follicular growth, but increased sensitivity was noted only for LH. The development of granulosa cell capacity to secrete P appears to be more advanced in 5-day rats than in 4-day rats relative to the next estrus. Because T synergized with LH and FSH to increase P secretion without altering sensitivity to gonadotropins, it probably acts at a site distal to gonadotropin receptors.