Ana Simonović

Biosynthesis and localization of parthenolide in glandular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.)

Feverfew (Tanacetum parthenium) is a perennial medicinal herb and is a rich source of sesquiterpene lactones. Parthenolide is the main sesquiterpene lactone in feverfew and has attracted attention because of its medicinal potential for treatment of migraine and cancer. In the present work the parthenolide content in different tissues and developmental stages of feverfew was analyzed to study the timing and localization of parthenolide biosynthesis. The strongest accumulating tissue was subsequently used to isolate sesquiterpene synthases with the goal to isolate the gene encoding the first dedicated step in parthenolide biosynthesis. This led to the isolation and charachterization of a germacrene A synthase (TpGAS) and an (E)-β-caryophyllene synthase (TpCarS). Transcript level patterns of both sesquiterpene synthases were analyzed in different tissues and glandular trichomes. Although TpGAS was expressed in all aerial tissues, the highest expression was observed in tissues that contain high concentrations of parthenolide and in flowers the highest expression was observed in the biosynthetically most active stages of flower development. The high expression of TpGAS in glandular trichomes which also contain the highest concentration of parthenolide, suggests that glandular trichomes are the secretory tissues where parthenolide biosynthesis and accumulation occur.

Reverse Transcription of 18S rRNA with Poly(dT)18 and Other Homopolymers

Ribosomal 18S RNA is widely used as a housekeeping gene in expression studies, including end-point PCR, Northern analysis, and real-time experiments. However, there are two disadvantages and two points of error introduction in using 18S rRNA as a reference gene. First, 18S has no poly(A) tail, so it is commonly reverse transcribed with specific primers or random hexamers, independently from poly(dT)-primed transcripts. Secondly, due to its abundance, the 18S cDNA must be extensively diluted to be comparable to the tested genes. In this study, 18S rRNA from five taxonomically diverse plant species, including Physcomitrella patens, Adiantum capillus-veneris, Centaurium erythraea, Arabidopsis thaliana, and Zea mays, was successfully reverse transcribed (RT) using poly(dT)18. As all other homopolymers, including poly(dA)18, poly(dC)18, and poly(dG)18, could serve as RT primers, it was concluded that homopolymers anneal by mispriming at the sites of complementary homopolymeric runs or segments rich in complementary base. Poly(dC)18 was the most efficient as RT primer, and the only one which interfered with subsequent PCR, giving species-specific pattern of products. Poly(dT)-primed RT reactions were less efficient in comparison to specific primer or random hexamer-primed reactions. Homopolymeric priming of 18S in RT reactions is general in terms of RNA origin and the method of RNA isolation and is possibly applicable to other tailless housekeeping genes.

Hairy root exudates of allelopathic weed Chenopodium murale L. induce oxidative stress and down-regulate core cell cycle genes in Arabidopsis and wheat seedlings

The effects of Chenopodium murale root exudates, applied as phytotoxic medias (PMs), were tested on Arabidopsis thaliana and Triticum aestivum. The effects of PMs, where wild-type roots (K), hairy roots derived from roots (R clones) or from cotyledons (C clones) were cultured, were different. K medium suppressed Arabidopsis germination, while other PMs reduced root and leaf elongation and the number of rosette leaves. R media were more phytotoxic than C media. Treatment of Arabidopsis with R8 down-regulated expression of core cell cycle genes: cyclin-dependent kinase (CDK) A1;1, four B-class CDKs, and cyclins CYCA3;1, CYCB2;4, CYCD4;2 and CYCH1 in root and shoot tips. Only CYCD2;1 transcript was elevated in treated shoots, but down-regulated in roots. Wheat Ta-CDC2 and Ta-CYCD2 genes showed the same expression profiles as their Arabidopsis counterparts, CDKA1;1 and CYCD2;1. PMs also caused increase of antioxidative enzyme activities in both plants. Exposure of Arabidopsis to PMs induced one catalase isoform, but repressed another, resulting in no net change of catalase activity. Wheat seedlings treated with PMs had catalase activity significantly elevated in all treatments, particularly in shoots. In both plants, PMs induced the activity of different peroxidase isozymes and total peroxidase activity. Both plants responded to phytotoxic treatments by induction of CuZn-superoxide dismutase. Thus, the phytotoxicity of C. murale root exudates is, at least partially, based on down-regulation of the cell cycle regulators and on generation of oxidative stress in the affected plants. We propose that C. murale root exudates should be considered as means of biological weed control.

Cytokinin Profiles of AtCKX2-Overexpressing Potato Plants and the Impact of Altered Cytokinin Homeostasis on Tuberization In Vitro

Genes encoding cytokinin oxidase/dehydrogenase (CKX) enzymes have been used lately to study cytokinin homeostasis in a variety of plant species. In this study AtCKX2-overexpressing potato plants were engineered and grown in vitro as a model system to investigate the effects of altered cytokinin levels on tuber formation and tuber size. Protein extracts from shoots and roots of transformed potato plants exhibited higher CKX activity compared to control plants. Total endogenous cytokinin levels were generally not decreased in AtCKX2 overexpressors. However, levels of bioactive cytokinins were markedly lowered, which was accompanied by increased levels of O- and N-glucosides in some transgenic lines. The AtCKX2-overexpressing plants displayed reduced shoot growth but other symptoms of the “cytokinin deficiency syndrome” were not recorded. The transgenic plants were able to produce tubers in noninducing conditions. In inducing conditions they developed larger tubers than control. Tubers were also formed on a greater portion of the analyzed AtCKX2 plants, but with a lower number of tubers per plant compared to control. Taken together, our data suggest that cytokinins cannot be regarded simply as positive or negative regulators of tuberization, at least in vitro. Interactions with other plant hormones that play an important role in control of tuberization, such as gibberellins, should be further studied in detail.

Herbicide Phosphinothricin Causes Direct Stimulation Hormesis

Herbicide phosphinothricin (PPT) inhibits glutamine synthetase (GS), a key enzyme in nitrogen assimilation, thus causing ammonia accumulation, glutamine depletion and eventually plant death. However, the growth response of Lotus corniculatus L. plants immersed in solutions with a broad range of PPT concentrations is biphasic, with pronounced stimulating effect on biomass production at concentrations ≤ 50 μM and growth inhibition at higher concentrations. The growth stimulation at low PPT concentrations is a result of activation of chloroplastic isoform GS2, while the growth suppression is caused by inhibition of both cytosolic GS1 and GS2 at higher PPT concentrations. Since the results are obtained in cell-free system (e.g. protein extracts), to which the principles of homeostasis are not applicable, this PPT effect is an unambiguous example of direct stimulation hormesis. A detailed molecular mechanism of concentration-dependent interaction of both PPT and a related GS inhibitor, methionine sulfoximine, with GS holoenzymes is proposed. The mechanism is in concurrence with all experimental and literature data.

Characterization of natural leaf senescence in tobacco (Nicotiana tabacum) plants grown in vitro

Leaf senescence is a highly regulated final phase of leaf development preceding massive cell death. It results in the coordinated degradation of macromolecules and the subsequent nutrient relocation to other plant parts. Very little is still known about early stages of leaf senescence during normal leaf ontogeny that is not triggered by stress factors. This paper comprises an integrated study of natural leaf senescence in tobacco plants grown in vitro, using molecular, structural, and physiological information. We determined the time sequence of ultrastructural changes in mesophyll cells during leaf senescence, showing that the degradation of chloroplast ultrastructure fully correlated with changes in chlorophyll content. The earliest degenerative changes in chloroplast ultrastructure coinciding with early chromatin condensation were observed already in mature green leaves. A continuum of degradative changes in chloroplast ultrastructure, chromatin condensation and aggregation, along with progressive decrease in cytoplasm organization and electron density were observed in the course of mesophyll cells ageing. Although the total amounts of endogenous cytokinins gradually increased during leaf ontogenesis, the proportion of bioactive cytokinin forms, as well as their phosphate precursors, in total cytokinin content rapidly declined with ageing. Endogenous indole-3-acetic acid (IAA) levels were strongly reduced in senescent leaves, and a decreasing tendency was also observed for abscisic acid (ABA) levels. Senescence-associated tobacco cysteine proteases (CP, E.C. 3.4.22) CP1 and CP23 genes were induced in the initial phase of senescence. Genes encoding glutamate dehydrogenase (GDH, E.C. 1.4.1.2) and one isoform of cytosolic glutamine synthetase (GS1, E.C. 6.3.1.2) were induced in the late stage of senescence, while chloroplastic GS (GS2) gene showed a continuous decrease with leaf ageing.

Plant regeneration in leaf culture of Centaurium erythraea Rafn. Part 1: The role of antioxidant enzymes

Centaurium erythraea Rafn. is a medicinal plant rich in secoiridoids and xanthones and used for gastrointestinal disorders, fever, anemia and many other conditions. C. erythraea is characterized with extraordinary developmental plasticity and manageability in vitro; thus we propose it as an excellent experimental model system for studies in developmental biology. Hereby we describe regeneration of centaury from leaf explants that can proceed via somatic embryogenesis or organogenesis on inductive media containing 2,4-D and CPPU. In the absence of growth regulators, shoots and roots appeared without callusing, on light or in darkness, respectively. Indirect somatic embryogenesis was induced in the presence of growth regulators occurring both on light and in darkness. Light was obligatory for indirect shoot development, where adventitious buds formed simultaneously with somatic embryos. Dynamic changes of antioxidative activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) in response to morphogenetic changes were followed during developmental pathways in vitro. Wounding of centaury leaves immediately induced all SOD and CAT isoforms but caused a decrease in POX activity. In control leaves and leaf explants, three Cu/Zn-SOD activities were detected, which gradually decreased on inductive treatments on light, but remained unchanged during growth in darkness. Morphogenetic paths on all hormonal and light treatments where characterized with dynamic changes of CAT activity (comprised of three major CAT isoforms), but generally CAT was reduced during morphogenesis induction. POX activity was strongly induced during morphogenesis in all treatments.

Interaction of gibberellins and fusicoccin in growth retardant- and far red light-inhibited germination of lettuce seeds

Germination of lettuce seeds cv. May Queen iscompletely prevented either with 10 µM tetcyclacisor with continuous FR illumination. GA3 and the N-substituted phtalimide, AC 94,377, werepartially effective in overcoming tetcyclacis-inducedinhibition but ineffective on photoinhibited seeds. FCcompletely reversed tetcyclacis inhibition and inducedca. 60% germination in continuous FR light. Aninteraction between FC and GA3 (as well asbetween FC and AC 94,377) was evident in stimulationof germination under both inhibitory conditions.Interaction was calculated as a ratio of thepercentage of seeds germinated under the simultaneousaction of stimulators compared to their additiveeffect. This was 2.54 for tetcyclacis- and 2.95 forphotoinhibited seeds. It is concluded that thistype of interaction is promotive synergism. Themagnitude of the interaction was highest if theapplication of FC was delayed after GA3application, and the optimal time lag was 6 h fortetcyclacis-inhibited, or 24 h for photoinhibited seeds.