Anna Maria Nuutila

Comparison of antioxidant activities of onion and garlic extracts by inhibition of lipid peroxidation and radical scavenging activity

The antioxidant activities of the methanol extracts of selected varieties and parts of garlic and onion were determined by two methods: inhibition of lipid peroxidation induced by tert-butyl hydroperoxide in isolated rat hepatocytes and scavenging activity against diphenylpicrylhydrazyl radical. The total phenolics and the main flavonoids of the hydrolysed onion and garlic samples were also analysed. The antioxidant activities obtained by the two methods were compared. Both methods gave similar antioxidant activities for pure compounds and Allium extracts. However, the radical scavenging method had many benefits compared to the lipid peroxidation method, being easier, cheaper, more specific and reproducible. The radical scavenging activities also correlated positively with the total phenolics of the extracts. Onions had clearly higher radical scavenging activities than garlic, red onion being more active than yellow onion. The skin extracts of onion possessed the highest activities.

A functional genomics approach toward the understanding of secondary metabolism in plant cells

Despite the tremendous importance of secondary metabolites for humans as for the plant itself, plant secondary metabolism remains poorly characterized. Here, we present an experimental approach, based on functional genomics, to facilitate gene discovery in plant secondary metabolism. Targeted metabolite analysis was combined with cDNA-amplified fragment length polymorphism-based transcript profiling of jasmonate-elicited tobacco Bright yellow 2 cells. Transcriptome analysis suggested an extensive jasmonate-mediated genetic reprogramming of metabolism, which correlated well with the observed shifts in the biosynthesis of the metabolites investigated. This method, which in addition to transcriptome data also generates gene tags, in the future might lead to the creation of novel tools for metabolic engineering of medicinal plant systems in general.

Transgenic oat plants via visual selection of cells expressing green fluorescent protein

Abstract New selectable markers and selection systems are needed to increase the efficiency and flexibility of plant transformation. The objective of this research was to determine if the green fluorescent protein (gfp) gene could be utilized as a visual selectable marker for transformation of oat (Avena sativa L.). A modified gfp gene was delivered into oat cells by microprojectile bombardment. Cell clusters expressing gfp were visually identified using fluorescence microscopy and physically isolated at each subculture. Eleven independent transgenic cell lines were obtained, and fertile plants regenerated from all lines. Transgene integration and expression were confirmed in transgenic plants and progeny. Transgene expression segregated in a 3 : 1 ratio in progeny of the majority of the transgenic lines.

Glycosylated F4 (K88) fimbrial adhesin FaeG expressed in barley endosperm induces ETEC-neutralizing antibodies in mice

The F4-positive enterotoxigenic Escherichia coli (ETEC) strains are a frequent cause of porcine post-weaning diarrhea. Orally administered F4 fimbriae or FaeG, the major subunit and adhesin of F4, induce a protective mucosal immune response in F4 receptor-positive piglets. Feed plants carrying immunogenic subunit proteins can offer great advantages for oral vaccination of domestic animals. Here, we describe high-level endosperm-specific production (1% of total soluble proteins) of FaeG in the crop plant barley. The endoplasmic reticulum-targeted recombinant endospermic FaeG (erFaeG) was shown to be heterogeneously glycosylated. The erFaeG showed resistance at digestive conditions simulating piglet gastric fluid. Glycosylation did not abolish the immunogenic character of the FaeG protein, since erFaeG was able to induce F4 fimbria-specific antibodies in mice. Biological activity of these anti-F4 antibodies was demonstrated in vitro by blocking the attachment of the F4+ ETEC to the F4 receptors present on porcine intestinal enterocytes.

Heterologous Expression of Vitreoscilla Hemoglobin (VHb) and Cultivation Conditions Affect the Alkaloid Profile of Hyoscyamus muticus Hairy Roots

Fast‐growing hairy root cultures of Hyoscyamus muticus induced by Agrobacterium rhizogenes offer a potential production system for tropane alkaloids. Oxygen deficiency has been shown to limit growth and biomass accumulation of hairy roots, whereas little experimental data is available on the effect of oxygen on alkaloid production. We have investigated the effect of Vitreoscilla hemoglobin (VHb) expression and cultivation conditions on the complete alkaloid profile of H. muticus hairy roots in shake flasks and in a laboratory scale bioreactor. We optimized the growth medium composition and studied the effects of sucrose, ammonium, nitrate, and phosphate on growth and alkaloid production. Maximum biomass accumulation was achieved with the highest and maximum hyoscyamine content with the lowest sucrose concentration. The optimum nitrate concentration for growth was higher for the VHb line than the control. Neither VHb expression nor aeration improved the hyoscyamine content significantly, thus suggesting that hyoscyamine biosynthesis is not limited by oxygen availability. Interestingly, the effect of VHb expression on the alkaloid profile was slightly different from that of aeration. VHb expression did not affect the concentrations of cuscohygrine, which was increased by aeration. Therefore, the effect of VHb is probably not related only to its ability to increase the intracellular effective oxygen concentration.

Heterologous expression of Vitreoscilla haemoglobin in barley ( Hordeum vulgare )

The vhb gene encoding Vitreoscilla haemoglobin (VHb) was transferred to barley with the aim of studying the role of oxygen availability in germination and growth. Previous findings indicate that VHb expression improves the efficiency of energy generation during oxygen-limited growth, and germination is known to be an energy demanding growth stage during which the embryos also suffer from oxygen deficiency. When subjected to oxygen deficiency, the roots of vhb-expressing barley plants showed a smaller increase in alcohol dehydrogenase (ADH) activity than those of the control plants. This indicates that VHb plants experienced less severe oxygen deficiency than the control plants, possibly due to the ability of VHb to substitute ADH for recycling NADH and maintaining glycolysis. In contrast to previous findings, we found that constitutive vhb expression did not improve the germination rate of barley kernels in any of the conditions studied. In some cases, vhb expression even slowed down germination slightly. VHb production also appeared to restrict root formation in young seedlings. The adverse effects of VHb on germination and root growth may be related to its ability to scavenge nitric oxide (NO), an important signal molecule in both seed germination and root formation. Because NO has both cytotoxic and stimulating properties, the effect of vhb expression in plants may depend on the level and role of endogenous NO in the conditions studied. VHb production also affected the levels of endogenous barley haemoglobin, which may explain the relatively moderate effects of VHb in this study.

Inducing homozygosity in transgenic barley (Hordeum vulgare L.) by microspore culture

Homozygosity was induced in transgenic barley by microspore culture. Spikes of transgenic barley plants carrying microspores in the late uni-nucleate stage were cold pretreated. Teflon rod maceration and a density of 100 000 viable micropores per plate were used. The developed calli were regenerated and plantlets were treated with colchicine. The microspore culture of 16 mother plants (three transgenic lines) resulted in 927 green regenerants. Of these plants, 476 were transferred to soil, 380 were transgenic, 358 reached maturity and 350 were fertile with a normal seed-set carrying a yield of 6.9 kg. A production efficiency of 0.8 fertile transgenic doubled haploid barley plants per spike used for microspore isolation was recorded. The produced transgenic seeds were used in malting experiments.

Modifying seeds to produce proteins

Although seeds are mainly used for food or feed, they are also used as raw materials for various other industrial applications. Improvement of the nutritional quality of protein in legumes and cereals has been the prime aim of plant breeders. Crop breeding has undergone major changes during the last decades and new selection strategies are based on sophisticated genetics, the use of computational power, and biometric methods in field trials. On the other hand, the processing quality of seeds can be modified by altering the structural grain constituents or the enzyme activities that mobilize storage reserves of the seeds. Recent developments in the molecular biology of seed proteins offer breeders new tools for this work.

Secondary metabolites in the post-genomic era

Many important pharmaceutical compounds are still isolated from plants. Due to their complex structures the chemical synthesis of these secondary metabolites is usually not applied. Plant cell cultures would offer an attractive alternative production system. However, the yields in cell cultures have only in few cases been commercially feasible. Futhermore, due to the lack of understanding of the biosynthetic pathways, genetic engineering has not had much success until now. It is expected that the application of genomics, proteomics and metabolomics tools will create a paradigm shift in our ability to engineer the often complex biosynthetic pathways of plant secondary metabolites. To illustrate the power of this approach, we present some data on the genome-wide transcript cDNA-AFLP profiling in combination with a GC-MS-SIM alkaloid analysis of methyl jasmonate elicited BY-2 tobacco cells used as a model system.