The role of AtPP2-A3 and AtPP2-A8 genes encoding Nictaba-related lectin domains in the defense response of Arabidopsis thaliana to Heterodera schachtii.

MAIN CONCLUSION: Expression levels of AtPP2-A3 and AtPP2-A8 are reduced in syncytia induced by Heterodera schachtii and decline of their expression levels decreases host susceptibility, whereas their overexpression promotes susceptibility to parasite. Plant-parasitic nematodes cause huge crop losses worldwide. Heterodera schachtii is a sedentary cyst-forming nematode that induces a feeding site called a syncytium via the delivery of secreted chemical substances (effectors) to host cells, which modulate host genes expression and phytohormone regulation patterns. Genes encoding the Nictaba-related lectin domain have been found among the plant genes with downregulated expression during the development of syncytia induced by H. schachtii in Arabidopsis thaliana roots. To investigate the role of two selected Nictaba-related genes in the plant response to beet cyst nematode parasitism, mutants and plants overexpressing AtPP2-A3 or AtPP2-A8 were infected, and promoter activity and protein localization were analyzed. In wild-type plants, AtPP2-A3 and AtPP2-A8 were expressed only in roots, especially in the cortex and rhizodermis. After nematode infection, their expression was switched off in regions surrounding a developing syncytium. Astonishingly, plants overexpressing AtPP2-A3 or AtPP2-A8 were more susceptible to nematode infection than wild-type plants, whereas mutants were less susceptible. Based on these results and changes in AtPP2-A3 and AtPP2-A8 expression patterns after treatments with different stress phytohormones, we postulate that the AtPP2-A3 and AtPP2-A8 genes play important roles in the defense response to beet cyst nematode infection.

Arabidopsis thaliana Myb59 Gene Is Involved in the Response to Heterodera schachtii Infestation, and Its Overexpression Disturbs Regular Development of Nematode-Induced Syncytia.

Transcription factors are proteins that directly bind to regulatory sequences of genes to modulate and adjust plants' responses to different stimuli including biotic and abiotic stresses. Sedentary plant parasitic nematodes, such as beet cyst nematode, Heterodera schachtii, have developed molecular tools to reprogram plant cell metabolism via the sophisticated manipulation of genes expression, to allow root invasion and the induction of a sequence of structural and physiological changes in plant tissues, leading to the formation of permanent feeding sites composed of modified plant cells (commonly called a syncytium). Here, we report on the AtMYB59 gene encoding putative MYB transcription factor that is downregulated in syncytia, as confirmed by RT-PCR and a promoter pMyb59::GUS activity assays. The constitutive overexpression of AtMYB59 led to the reduction in A. thaliana susceptibility, as indicated by decreased numbers of developed females, and to the disturbed development of nematode-induced syncytia. In contrast, mutant lines with a silenced expression of AtMYB59 were more susceptible to this parasite. The involvement of ABA in the modulation of AtMYB59 gene transcription appears feasible by several ABA-responsive cis regulatory elements, which were identified in silico in the gene promoter sequence, and experimental assays showed the induction of AtMYB59 transcription after ABA treatment. Based on these results, we suggest that AtMYB59 plays an important role in the successful parasitism of H. schachtii on A. thaliana roots.

The Interactions of Nintedanib and Oral Anticoagulants-Molecular Mechanisms and Clinical Implications.

Nintedanib is a synthetic orally active tyrosine kinase inhibitor, whose main action is to inhibit the receptors of the platelet-derived growth factor, fibroblast growth factor and vascular endothelial growth factor families. The drug also affects other kinases, including Src, Flt-3, LCK, LYN. Nintedanib is used in the treatment of idiopathic pulmonary fibrosis, chronic fibrosing interstitial lung diseases and lung cancer. The mechanism of action suggests that nintedanib should be considered one of the potential agents for inhibiting and revising the fibrosis process related to COVID-19 infections. Due to the known induction of coagulation pathways during COVID-19 infections, possible interaction between nintedanib and anticoagulant seems to be an extremely important issue. In theory, nintedanib could increase the bleeding risk, thrombosis and lead to thrombocytopenia. The data from clinical trials on the concomitant use of nintedanib and antithrombotic agents is very limited as this patient group was within the standard exclusion criteria. Nintedanib is an important therapeutic option, despite its interaction with anticoagulants. If anticoagulant therapy is necessary, the more effective and safer option is the concomitant administration of DOACs and nintedanib, especially when drug-monitored therapy will be used in patients at high risk of bleeding complications.

Nitric Oxide-Induced Dormancy Removal of Apple Embryos Is Linked to Alterations in Expression of Genes Encoding ABA and JA Biosynthetic or Transduction Pathways and RNA Nitration.

Short-term (3 h) treatment of embryos isolated from dormant apple (Malus domestica Borkh.) seeds with NO donors stimulates their transition from dormancy to germination. Seed dormancy is maintained by ABA, while germination is controlled mainly by gibberellins (GAs) and jasmonic acid (JA). NO-induced dormancy removal correlates with low ABA concentration in embryonic axes and reduced embryo sensitivity to ABA. We analyzed the expression of genes encoding key enzymes of ABA degradation (CYP707A1, CYP707A2), biosynthesis (NCED3, NCED9), and elements of the ABA transduction pathway (PYL1, PYL2, RCAR1, RCAR3, PP2CA, ABI1, ABI2, SNRK2, ABI5, AREB3, ABF). A role for JA in the regulation of germination led us to investigate the expression of genes encoding enzymes of JA biosynthesis (AOS1, JMT, JAR1) and the transduction pathway (COI1, MYC2, JAZ3, JAZ12). The expression profiles of the genes were estimated in embryonic axes isolated from dormant or NO fumigated apple embryos. The analyzed genes were differentially regulated during dormancy alleviation, the main modifications in the transcription level were detected for NCED3, NCED9, CYP707A2, RCAR1, ABF, AOS1, JMT, JAR1 and JAZ3. A regulatory role of NO in the removal of seed dormancy is associated with the stimulation of expression of genes related to ABA degradation, down-regulation of genes responsible for ABA synthesis, an increase of expression level of genes engaged in JA synthesis and modification of the expression of genes engaged in signaling pathways of the hormones. To confirm a signaling role of NO during dormancy breakage, an increased RNA nitration level in embryonic axes was demonstrated.

Mutations in Selected ABA-Related Genes Reduce Level of Arabidopsis thaliana Susceptibility to the Beet Cyst Nematode Heterodera schachtii.

Heterodera schachtii is a common parasite of many important crops such as beets and Brassicaceae (oilseed rape, cabbage or mustard). Arabidopsis thaliana is a model plant also used for studying defence responses to pathogens or pest infections. Defence responses of plants are often regulated and fine-tuned by stress phytohormones: salicylic acid (SA), jasmonic acid (JA), ethylene (Et) and abscisic acid (ABA), of which the role of ABA in these responses is the least examined. The aim of this study was to show, if and which genes related to ABA turnover can be modulated during the development of nematode-induced feeding sites in A. thaliana roots. To answer the question, we performed infection tests on wild type and ABA mutant roots and analysed the expression levels of selected ABA-related genes (ABI1, ABI2, ABI5, PYL5, PYL6, CYP707A1 and CYP707A4) at the early stage of root infection. Our results show that the expression of ABI2, ABI5 (ABA signalling) and CYP707A4 (ABA metabolism) genes was upregulated in feeding sites at 4 dpi, whereas the level of expression of PYL5 and PYL6 (ABA receptors) genes was decreased. Mutations in ABI1, ABI2, ABI5, CYP707A1 or CYP707A4 genes led to a decrease of A. thaliana susceptibility verbalised as the number of fully developed females, whereas mutations in PYL5 or PYL6 genes did not influence the number of females of the nematode. Based on the results, it can be concluded that the modifications of analysed ABA-related gene expression are required for the proper development of nematodes; however, further in-depth analyses are required.

Inhibition of tomato (Solanum lycopersicum L.) root growth by cyanamide is due to altered cell division, phytohormone balance and expansin gene expression.

Cyanamide (CA) has been reported as a natural compound produced by hairy vetch (Vicia villosa Roth.) and it was shown also to be an allelochemical, responsible for strong allelopathic potential in this species. CA phytotoxicity has been demonstrated on various plant species, but to date little is known about its mode of action at cellular level. Treatment of tomato (Solanum lycopersicum L.) roots with CA (1.2 mM) resulted in inhibition of growth accompanied by alterations in cell division, and imbalance of plant hormone (ethylene and auxin) homeostasis. Moreover, the phytotoxic effect of CA was also manifested by modifications in expansin gene expression, especially in expansins responsible for cell wall remodeling after the cytokinesis (LeEXPA9, LeEXPA18). Based on these results the phytotoxic activity of CA on growth of roots of tomato seedlings is likely due to alterations associated with cell division.

Arabidopsis thaliana AtHRS1 gene is involved in the response to Heterodera schachtii infection and its overexpression hampers development of syncytia and involves a jasmonic acid-dependent mechanism.

Sedentary plant parasitic nematodes have developed competences to reprogram host plant cell metabolism via sophisticated manipulation of gene expression, leading to the formation of permanent feeding sites for an unlimited source of food. Arabidopsis thaliana and the beet cyst nematode Heterodera schachtii is a good model for studying the mechanisms of compatible plant-nematode interactions and basic plant responses to nematode infection. Transcription factors are proteins that modulate plant reactions during regular development and under different biotic and abiotic stresses via direct binding to promoter regions of genes. Here, we report on the AtHRS1 gene encoding a MYB-related transcription factor belonging to the GARP family, whose expression is downregulated in syncytia, as confirmed by gene expression analysis. Constitutive overexpression of AtHRS1 disturbed the development of nematode-induced syncytia and led to a reduction in the number of developed females in transgenic A. thaliana roots. In contrast, the hrs1 mutant with decreased expression of AtHRS1 was more susceptible to cyst nematode infection. The influence of AtHRS1 on selected elements of the JA-dependent defence pathway suggests its mode of action in plant response to nematode attack. Based on these results, we suggest that the downregulation of AtHRS1 expression by nematode is important for its successful development.

Oral versus intravenous hydration and renal function in diabetic patients undergoing percutaneous coronary interventions.

BACKGROUND: Contrast-induced nephropathy (CIN) is a serious complication of percutenous coronary interventions (PCI). Proper hydration reduces the risk of PCI. Wheter oral hydration is as effective as intravenous one has not been well established. AIM: To determine the effects of oral hydration with mineral water versus intravenous hydration with isotonic solution (0.9% NaCl) on renal function in diabetic patients undergoing coronary angiography and angioplasty. METHODS: The study included 102 patients (age 67 ± 7.8 years, 44 female/58 male). Eligible patients (group 1 - 52 pts) were hydrated intravenously (1 mL/kg/h) 6 hours before and during 12 hours following PCI with isotonic solution (0.9% NaCl). Fifty patients (group 2) were randomised to receive oral mineral water (1 mL/kg/h) 6-12 hours before and during 12 hours following angiography or angioplasty. All patients during the procedure received contrast medium ioversol. Primary endpoint of the study was the evaluation of renal function before and 72 hours after contrast medium administration. RESULTS: Baseline creatinine clearance was 70.3 ± 21.22 mL/min in group 1 and 78.69 ± 19.92 mL/min in group 2 (NS). The mean volume of contrast medium was 101.1 ± 36.7 mL in group 1 and 110.4 ± 45.3 mL in group 2 (NS). At 72 hours after the procedure, creatinine clearance was 65.3 ± 23.39 mL/min in group 1 and 73.5 ± 21.94 mL/min in group 2 (NS). CONCLUSIONS: Our study demonstrates that the oral hydration with mineral water and intravenous hydration with 0.9% NaCl have similar effects on renal function in diabetic patients undergoing coronary angiography and angioplasty.

[Cytochrome P450 isoenzymes in metabolism of endo- and exogenic compounds]. / Izoenzymy cytochromu P450 w metabolizmie zwiazków endo- i egzogennych.

Cytochrome P450 enzymes (CYPs) belong to hemeproteins found in all living organisms. In eucaryotic cells they are responsible for biosynthesis and transformations of endogenic lipids as well as for the metabolism of xenobiotics, including therapeutic agents. C-Oxidation (hydroxylation, epoxydation, peroxydation), N-oxidation and S-oxidation as well as oxidative O-, S-, and N-dealkylation of substrates are catalysed by CYPs. These monooxygenation reactions sometimes result in dimerisation, isomerisation or cyclisation of the substrate. Human cytochrome P450 isoenzymes are described by 57 genes and products of their expression are different in specificity. For instance CYP51A1 is crucial for sterol biosynthesis, whereas CYP7A1, 7B1 and 39A1 take part in synthesis of bile acids and CYP46A1 in metabolism of cholesterol. Therapeutic agents are metabolised mainly by CYP3A4, 2D6, 2C9 and 2C19. In addition, CYP2E1 takes part in metabolism of ethyl alcohol and CYP1A1/2 in activation of carcinogens. Metabolism of xenobiotics seems to be the defence mechanism against toxic effects of strange chemicals, whereas, it is also the way of drug activation and detoxication.

[NADPH-cytochrome P450 reductase, not only the partner of cytochrome P450]. / Reduktaza NADPH: cytochrom P450--nie tylko partner cytochromu P450.

NADPH-cytochrome P450 reductase, CPR, the enzyme of the majority of eucaryotic cells belongs to the family of diflavin reductases and is usually located in endoplasmic reticulum. This protein is build of three domains. The first one, C-terminal, binds FAD and NADPH, the second one, N-terminal, binds FMM, whereas the third one is the regulatory domain. Catalytic cycle of the enzyme runs by intermediate FMNH-FADH with the participation of conformational changes induced by NADPH binding to the active centre of the enzyme. It has been shown in mice that CPR was necessary for the action of cytochrome P450 monooxygenase system, but this system is not crucial for animal surviving. CPR participates also in electron transport to cytochrome b5, heme oxidase, squalen monooxygenase and 7-dehydrocholesterole reductase. Furthermore, its own crucial task is the catalysis of reductive metabolism of prodrugs, particularly antitumor agents.

[Polymorphism and the level of P450 gene expression in xenobiotic metabolism]. / Rola polimorfizmu i zróznicowanej ekspresji genów cytochromów P450 w metabolizmie ksenobiotyków.

Polymorphism and the level of P450 gene expression influence metabolic pathway of xenobiotics. The highest number, over 40 polymorphic forms were determined for CYP2D6 enzyme and they expressed various activity. Whereas, only two forms of lower activity were found in the case of CYP2C9. However, CYP3A4 mutations have not influenced metabolic activity of the enzyme. Three transcription factors PXR, CAR and AhR are crucial for gene expression regulation of cytochrome P450. They regulate the induction of CYP3A4, CYP2B6 and CYP1A2 genes, respectively. PXR is activated by binding specific ligand, whereas the activator of constitutive CAR receptor seems not to be the ligand and it might induce dephosphorylation. After being transported to the nucleus, activated receptors PXR, CAR or AhR formed heterodimers and then, with participation of the following coactivators bind to PBREM, XREM or DRE regulatory motifs of DNA, respectively. The activation of nuclear receptors strongly influences the drug-drug interactions that are essential for the efficiency of drug, particularly in the field of anticancer therapy.

Fodder beet is a reservoir of drought tolerance alleles for sugar beet breeding.

Drought leads to serious yield losses and followed by increasing food prices. Thereby, drought tolerance is one of most important, pivotal issues for plant breeding and is determined by the very complex genetic architecture, which involves a lot of genes engaged in many cell processes. Within genomes of currently cultivated sugar beet forms, the number of favourable allelic variants is limited. However, there is a potential to identify genes related to drought tolerance deposited in genomes of wild or fodder relatives. Therefore, the goal of our study, was to identify the source of allelic variants involved in drought tolerance using a large spectrum of sugar or fodder beets and their wild relatives for analyses. Based on the drought tolerance index, calculated for morphophysiological traits, it was demonstrated that some of selected fodder beets showed the highest level of drought tolerance. The most drought tolerant fodder beet genotype did not show differences in the level of expression of genes engaged in osmoprotection and the antioxidative system, between control and drought condition, compared to sugar and wild beets. The genetic distance between selected beet forms was broad and ranged from 18 to 87%, however the most drought tolerant sugar, fodder and wild beets showed high genetic similarity and formed the common clade. Based on obtained results we propose that an adequate broad source of genes related to drought tolerance occurs in fodder beets, the crossing with which is easier, less time-consuming and more cost-effective than with wild forms of beets.

Growth performance, selected blood biochemical parameters and body weights of pre-pubertal gilts fed diets supplemented with different doses of zearalenone (ZEN).

The aim of this study was to determine whether exposure to low doses of zearalenone (ZEN) induces changes in the serum biochemical profile and body weights (BW). Pre-pubertal gilts (with BW of up to 14.5 kg) were administered ZEN in daily doses of 5 µg/kg BW (group 1, n = 15), 10 µg/kg BW (group 2, n = 15), 15 µg/kg BW (group 3, n = 15) or placebo (control group C, n = 15) throughout the experiment. Blood was sampled for analysis on 10 dates (at five-day intervals). Minor but statistically significant differences in the analysed serum biochemical parameters (ALT, AST, ALP, total cholesterol, total bilirubin, glucose, total protein, iron, BUN and urea) were observed in the studied groups. The biochemical parameters of the analysed gilts indicate that the maintenance of homeostasis and biotransformation of ZEN require considerable energy expenditure. Beginning on the fourth analytical date, BW gains were consistently higher in the experimental groups than in group C. The observed decrease in glucose and total protein levels can probably be attributed to higher BW gains and the ongoing ZEN biotransformation processes in the enterocytes and the liver.

Metabolic transformations of antitumor imidazoacridinone, C-1311, with microsomal fractions of rat and human liver.

The imidazoacridinone derivative C-1311 is an antitumor agent in Phase II clinical trials. The molecular mechanism of enzymatic oxidation of this compound in a peroxidase model system was reported earlier. The present studies were performed to elucidate the role of rat and human liver enzymes in metabolic transformations of this drug. C-1311 was incubated with different fractions of liver cells and the reaction mixtures were analyzed by RP-HPLC. We showed that the drug was more sensitive to metabolism with microsomes than with cytosol or S9 fraction of rat liver cells. Incubation of C-1311 with microsomes revealed the presence of four metabolites. Their structures were identified as dealkylation product, M0, as well as a dimer-like molecule, M1. Furthermore, we speculate that the hydroxyl group was most likely substituted in metabolite M3. It is of note that a higher rate of transformation was observed for rat than for human microsomes. However, the differences in metabolite amounts were specific for each metabolite. The reactivity of C-1311 with rat microsomes overexpressing P450 isoenzymes, of CYP3A and CYP4A families was higher than that with CYP1A and CYP2B. Moreover, the M1 metabolite was selectively formed with CYP3A, whereas M3 with CYP4A. In conclusion, this study revealed that C-1311 varied in susceptibility to metabolic transformation in rat and human cells and showed selectivity in the metabolism with P450 isoenzymes. The obtained results could be useful for preparing the schedule of individual directed therapy with C-1311 in future patients.

Tissue-culture-responsive and autotetraploidy-responsive changes in metabolic profiles of cucumber (Cucumis sativus L.).

Somaclonal variation commonly occurs during in vitro plant regeneration and may introduce unintended changes in numerous plant characters. In order to assess the range of tissue-culture-responsive changes on the biochemical level, the metabolic profiles of diploid and tetraploid cucumber R1 plants regenerated from leaf-derived callus were determined. Gas chromatography and mass spectrometry were used for monitoring of 48 metabolites and many significant changes were found in metabolic profiles of these plants as compared to a seed-derived control. Most of the changes were common to diploids and tetraploids and were effects of tissue culture. However, tetraploids showed quantitative changes in 14 metabolites, as compared to regenerated diploids. These changes include increases in serine, glucose-6P, fructose-6P, oleic acid and shikimic acid levels. Basing on this study we conclude that the variation in metabolic profiles does not correlate directly with the range of genome changes in tetraploids.

Suppression of NGB and NAB/ERabp1 in tomato modifies root responses to potato cyst nematode infestation.

Plant-parasitic nematodes cause significant damage to major crops throughout the world. The small number of genes conferring natural plant resistance and the limitations of chemical control require the development of new protective strategies. RNA interference or the inducible over-expression of nematicidal genes provides an environment-friendly approach to this problem. Candidate genes include NGB, which encodes a small GTP-binding protein, and NAB/ERabp1, which encodes an auxin-binding protein, which were identified as being up-regulated in tomato roots in a transcriptome screen of potato cyst nematode (Globodera rostochiensis) feeding sites. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization confirmed the localized up-regulation of these genes in syncytia and surrounding cells following nematode infection. Gene-silencing constructs were introduced into tomato, resulting in a 20%-98% decrease in transcription levels. Nematode infection tests conducted on transgenic plants showed 57%-82% reduction in the number of G. rostochiensis females in vitro and 30%-46% reduction in pot trials. Transmission electron microscopy revealed a deterioration of cytoplasm, and degraded mitochondria and plastids, in syncytia induced in plants with reduced NAB/ERabp1 expression. Cytoplasm in syncytia induced in plants with low NGB expression was strongly electron translucent and contained very few ribosomes; however, mitochondria and plastids remained intact. Functional impairments in syncytial cytoplasm of silenced plants may result from NGB's role in ribosome biogenesis; this was confirmed by localization of yellow fluorescent protein (YFP)-labelled NGB protein in nucleoli and co-repression of NGB in plants with reduced NAB/ERabp1 expression. These results demonstrate that NGB and NAB/ERabp1 play important roles in the development of nematode-induced syncytia.

Xyloglucan endotransglucosylase/hydrolase genes in cucumber (Cucumis sativus) - differential expression during somatic embryogenesis.

Defined changes in the cell wall directed by many proteins accompany every morphogenetic process in plants. Xyloglucan endotransglucosylase/hydrolase proteins (XTH; EC 2.4.1.207) have the potential to modify the hemicellulose matrix within the cell wall. Cs-XTH1 and Cs-XTH3 genes, which encode XTH proteins, were found among numerous genes that are differentially expressed after the induction of cucumber somatic embryogenesis. The expression of these genes increased during somatic embryogenesis. The Cs-XTH1 gene was localized on the second chromosome near the centromere region, whereas Cs-XTH3 was found in the middle of the fifth chromosome's longer arm. Northern blot hybridization showed that both genes were preferentially expressed in roots. We also observed higher accumulation of both transcripts in somatic embryos than in the proembryogenic mass. The localization of mRNA by in situ hybridization revealed that the Cs-XTH1 transcripts were largely accumulated in the presumptive cotyledon primordia of somatic embryos. The XTH gene family consists of a number of genes with a high degree of structural similarity. Screening a cucumber genomic library has identified other members of this gene family. The intron/exon structure, sequence similarities and the close chromosomal distance between some members suggest their common evolutionary origin. The involvement of XTH-related genes in somatic embryo formation is discussed.

Assessment of the physiological responses to drought in different sugar beet genotypes in connection with their genetic distance.

Drought affects many physiological processes, which influences plant productivity. The aim of this study was to evaluate the degree of genotypic diversity in drought tolerance of sugar beet genotypes (Beta vulgaris L.) in connection with their genetic distance. Three hybrid genotypes produced by crossing double haploid genotype (P-pollinator) with cytoplasmic male-sterile female part (MS), as well as with two parent lines, were examined. Drought conditions were imposed by the cessation of watering at the 3-4 leaf stage for about three months, after which irrigation was resumed. Control plants were optimally irrigated throughout the entire vegetation period. Long-term drought significantly increased the wilting of leaves (Wilt.), specific leaf weight (SLW), the succulence index (Suc.I), leaf senescence and membrane damage (El-l). Simultaneously, the osmotic potential (ψs), leaf area index (LAI), absorption of photosynthetic active radiation (PAR) and the efficiency of the photosynthetic apparatus (Φ PSII) declined under water deficit conditions. The examined genotypes demonstrated a clear diversity in their physiological response to drought. Based on these findings, we suggest that traits that are strongly correlated with root and sugar yield, e.g. Φ PSII, LAI, PAR absorption and ψs, could be used as potential selection criteria in physiological-associated breeding strategies to improve drought tolerance in sugar beet. There was not a significant correlation between the genetic distance separating different sugar beet genotypes and the observed heterotic effect of root or sugar yields, with the exception of heterosis of root yield under optimal conditions, where the correlation was negative.

Production of oleanolic acid glycosides by hairy root established cultures of Calendula officinalis L.

In order to initiate hairy root culture initiation cotyledons and hypocotyls of Calendula officinalis L. were infected with Agrobacterium rhizogenes strain ATCC 15834 or the same strain containing pCAMBIA 1381Z vector with ß-glucuronidase reporter gene under control of promoter of NIK (Nematode Induced Kinase) gene. The efficiency of induction of hairy roots reached 33.8% for cotyledons and 66.6% for hypocotyls together for both transformation experiments. Finally, eight control and nine modified lines were established as a long-term culture. The hairy root cultures showed the ability to synthesize oleanolic acid mainly (97%) as glycosides; control lines contained it at the average 8.42 mg · g(-1) dry weight in tissue and 0.23 mg · dm(-3) in medium; modified lines: 4.59 mg · g(-1) for the tissue, and 0.48 mg · dm(-3) for the medium. Additionally lines showed high positive correlation between dry/fresh weight and oleanolic acid concentration in tissue. Using the Killiani mixture in acidic hydrolysis of oleanolic acid glycosides released free aglycones that were partially acetylated in such conditions.

Identification of genes up-regulated during somatic embryogenesis of cucumber.

Somatic embryogenesis is a method of plant regeneration, but it can also be used as a model to study plant development. A normalized library of cDNA fragments representing genes up-regulated after the induction of somatic embryogenesis in cucumber suspension cultures was constructed using the suppression subtractive hybridization technique. Candidate cDNA fragments (119) were classified according to their similarity to genes encoding known proteins and the presence of potential functional domains. Of the translation products with homology to known proteins, about 23% were possibly involved in metabolism, 13% represented proteins with a probable role in cellular communication and signal transduction, about 12% were likely to participate in protein synthesis, while around 10% were potential transcription factors. The genes corresponding to four of the cDNAs were subsequently analyzed in more detail: CsSEF2, CsSEM1 and CsSESTK1 encoding putative transcription factors or co-activators, and CsSECAD1 encoding cinnamyl alcohol dehydrogenase. Full-length cDNAs were isolated and analyzed. RT-PCR confirmed the up-regulation of these genes after the induction of somatic embryogenesis and showed the presence of their transcripts in other tissues. The in situ localization of transcripts of the CsSEF2 and CsSEM1 genes demonstrated that signalling in somatic embryo tissues involving these factors is concentrated in the cotyledon primordia and roots.