A Critical Look at Prebiotics Within the Dietary Fiber Concept.

This article reviews the current knowledge of the health effects of dietary fiber and prebiotics and establishes the position of prebiotics within the broader context of dietary fiber. Although the positive health effects of specific fibers on defecation, reduction of postprandial glycemic response, and maintenance of normal blood cholesterol levels are generally accepted, other presumed health benefits of dietary fibers are still debated. There is evidence that specific dietary fibers improve the integrity of the epithelial layer of the intestines, increase the resistance against pathogenic colonization, reduce the risk of developing colorectal cancer, increase mineral absorption, and have a positive impact on the immune system, but these effects are neither generally acknowledged nor completely understood. Many of the latter effects are thought to be particularly elicited by prebiotics. Although the prebiotic concept evolved significantly during the past two decades, the line between prebiotics and nonprebiotic dietary fiber remains vague. Nevertheless, scientific evidence demonstrating the health-promoting potential of prebiotics continues to accumulate and suggests that prebiotic fibers have their rightful place in a healthy diet.

Wheat bran extract alters colonic fermentation and microbial composition, but does not affect faecal water toxicity: a randomised controlled trial in healthy subjects.

Wheat bran extract (WBE), containing arabinoxylan-oligosaccharides that are potential prebiotic substrates, has been shown to modify bacterial colonic fermentation in human subjects and to beneficially affect the development of colorectal cancer (CRC) in rats. However, it is unclear whether these changes in fermentation are able to reduce the risk of developing CRC in humans. The aim of the present study was to evaluate the effects of WBE on the markers of CRC risk in healthy volunteers, and to correlate these effects with colonic fermentation. A total of twenty healthy subjects were enrolled in a double-blind, cross-over, randomised, controlled trial in which the subjects ingested WBE (10 g/d) or placebo (maltodextrin, 10 g/d) for 3 weeks, separated by a 3-week washout period. At the end of each study period, colonic handling of NH3 was evaluated using the biomarker lactose[15N, 15N']ureide, colonic fermentation was characterised through a metabolomics approach, and the predominant microbial composition was analysed using denaturing gradient gel electrophoresis. As markers of CRC risk, faecal water genotoxicity was determined using the comet assay and faecal water cytotoxicity using a colorimetric cell viability assay. Intake of WBE induced a shift from urinary to faecal 15N excretion, indicating a stimulation of colonic bacterial activity and/or growth. Microbial analysis revealed a selective stimulation of Bifidobacterium adolescentis. In addition, WBE altered the colonic fermentation pattern and significantly reduced colonic protein fermentation compared with the run-in period. However, faecal water cytotoxicity and genotoxicity were not affected. Although intake of WBE clearly affected colonic fermentation and changed the composition of the microbiota, these changes were not associated with the changes in the markers of CRC risk.

Tolerance and the effect of high doses of wheat bran extract, containing arabinoxylan-oligosaccharides, and oligofructose on faecal output: a double-blind, randomised, placebo-controlled, cross-over trial.

Wheat bran extract (WBE) is a food-grade soluble fibre preparation that is highly enriched in arabinoxylan-oligosaccharides. In this placebo-controlled cross-over human intervention trial, tolerance to WBE as well as the effects of WBE on faecal parameters, including faecal output and bowel habits, were studied. After a 2-week run-in period, twenty healthy volunteers consumed WBE (15 g/d in the first week, 30 g/d in the second week), oligofructose (15 g/d in the first week, 30 g/d in the second week) and placebo (for 2 weeks) in a random order, with 2-week washout periods between each treatment period. Subjects collected a 72 h stool sample for analysis of faecal output, stool pH and stool moisture concentration. Additionally, the volunteers completed questionnaires scoring occurrence frequency and distress severity of eighteen gastrointestinal (GI) symptoms. An overall GI symptom measure was calculated to analyse the overall effect of WBE and oligofructose on GI symptoms. Intake of both 30 g/d WBE and 30 g/d oligofructose lowered stool pH, indicative of increased colonic fermentation, and increased stool moisture concentration as compared with placebo intake. Intake of 30 g/d oligofructose increased the overall GI symptom measure by 1·9-fold as compared with placebo intake. Intake of WBE at doses up to 30 g/d did not affect the overall GI symptom measure. WBE exerts beneficial effects on stool characteristics and is well tolerated at up to 30 g/d. Oligofructose exerts comparable beneficial effects on stool characteristics. However, intake of 30 g/d oligofructose appears to cause GI discomfort to some extent.

Effects of wheat bran extract containing arabinoxylan oligosaccharides on gastrointestinal parameters in healthy preadolescent children.

OBJECTIVES: We assessed whether wheat bran extract (WBE) containing arabinoxylan-oligosaccharides (AXOS) elicited a prebiotic effect and modulated gastrointestinal (GI) parameters in healthy preadolescent children upon consumption in a beverage. METHODS: This double-blind randomized placebo-controlled crossover trial evaluated the effects of consuming WBE at 0 (control) or 5.0 g/day for 3 weeks in 29 healthy children (8-12 years). Fecal levels of microbiota, short-chain fatty acids, branched-chain fatty acids, ammonia, moisture, and fecal pH were assessed at the end of each treatment and at the end of a 1-week run-in (RI) period. In addition, the subjects completed questionnaires scoring distress severity of 3 surveyed GI symptoms. Finally, subjects recorded defecation frequency and stool consistency. RESULTS: Nominal fecal bifidobacteria levels tended to increase after 5 g/day WBE consumption (P = 0.069), whereas bifidobacteria expressed as percentage of total fecal microbiota was significantly higher upon 5 g/day WBE intake (P = 0.002). Additionally, 5 g/day WBE intake induced a significant decrease in fecal content of isobutyric acid and isovaleric acid (P < 0.01), markers of protein fermentation. WBE intake did not cause a change in distress severity of the 3 surveyed GI symptoms (flatulence, abdominal pain/cramps, and urge to vomit) (P > 0.1). CONCLUSIONS: WBE is well tolerated at doses up to 5 g/day in healthy preadolescent children. In addition, the intake of 5 g/day exerts beneficial effects on gut parameters, in particular an increase in fecal bifidobacteria levels relative to total fecal microbiota, and reduction of colonic protein fermentation.

Effects of a wheat bran extract containing arabinoxylan oligosaccharides on gastrointestinal health parameters in healthy adult human volunteers: a double-blind, randomised, placebo-controlled, cross-over trial.

Wheat bran extract (WBE) is a food-grade soluble fibre preparation that is highly enriched in arabinoxylan oligosaccharides. In this placebo-controlled cross-over human intervention trial, tolerance and effects on colonic protein and carbohydrate fermentation were studied. After a 1-week run-in period, sixty-three healthy adult volunteers consumed 3, 10 and 0 g WBE/d for 3 weeks in a random order, with 2 weeks' washout between each treatment period. Fasting blood samples were collected at the end of the run-in period and at the end of each treatment period for analysis of haematological and clinical chemistry parameters. Additionally, subjects collected a stool sample for analysis of microbiota, SCFA and pH. A urine sample, collected over 48 h, was used for analysis of p-cresol and phenol content. Finally, the subjects completed questionnaires scoring occurrence frequency and distress severity of eighteen gastrointestinal symptoms. Urinary p-cresol excretion was significantly decreased after WBE consumption at 10 g/d. Faecal bifidobacteria levels were significantly increased after daily intake of 10 g WBE. Additionally, WBE intake at 10 g/d increased faecal SCFA concentrations and lowered faecal pH, indicating increased colonic fermentation of WBE into desired metabolites. At 10 g/d, WBE caused a mild increase in flatulence occurrence frequency and distress severity and a tendency for a mild decrease in constipation occurrence frequency. In conclusion, WBE is well tolerated at doses up to 10 g/d in healthy adults volunteers. Intake of 10 g WBE/d exerts beneficial effects on gut health parameters.

Consumption of breads containing in situ-produced arabinoxylan oligosaccharides alters gastrointestinal effects in healthy volunteers.

Arabinoxylan oligosaccharides (AXOS) are studied as food compounds with prebiotic potential. Here, the impact of consumption of breads with in situ-produced AXOS on intestinal fermentation and overall gastrointestinal characteristics was evaluated in a completely randomized, double-blind, controlled, cross-over study. Twenty-seven healthy volunteers consumed 180 g of wheat/rye bread with or without in situ-produced AXOS (WR(+) and WR(-), respectively) daily for 3 wk. Consumption of WR(+) corresponded to an AXOS intake of ~2.14 g/d. Refined wheat flour bread without AXOS (W(-)) (180 g/d) was provided during the 3-wk run-in and wash-out periods. At the end of each treatment period, participants collected urine for 48 h as well as a feces sample. Additionally, all participants completed a questionnaire about stool characteristics and gastrointestinal symptoms during the last week of each period. Urinary phenol and p-cresol excretions were significantly lower after WR(+) intake compared to WR(-). Consumption of WR(+) significantly increased fecal total SCFA concentrations compared to intake of W(-). The effect of WR(+) intake was most pronounced on butyrate, with levels 70% higher than after consumption of W(-) in the run-in or wash-out period. Consumption of WR(+) tended to selectively increase the fecal levels of bifidobacteria (P = 0.06) relative to consumption of W(-). Stool frequency increased significantly after intake of WR(+) compared to WR(-). In conclusion, consumption of breads with in situ-produced AXOS may favorably modulate intestinal fermentation and overall gastrointestinal properties in healthy humans.

Prebiotic effects and intestinal fermentation of cereal arabinoxylans and arabinoxylan oligosaccharides in rats depend strongly on their structural properties and joint presence.

SCOPE: Cereal arabinoxylan (AX) is one of the main dietary fibers in a balanced human diet. To gain insight into the importance of structural features of AX for their prebiotic potential and intestinal fermentation properties, a rat trial was performed. METHODS AND RESULTS: A water unextractable AX-rich preparation (WU-AX, 40% purity), water extractable AX (WE-AX, 81% purity), AX oligosaccharides (AXOS, 79% purity) and combinations thereof were included in a standardized diet at a 5% AX level. WU-AX was only partially fermented in the ceco-colon and increased the level of butyrate and of butyrate producing Roseburia/E. rectale spp. Extensive fermentation of WE-AX and/or AXOS reduced the pH, suppressed relevant markers of the proteolytic breakdown and induced a selective bifidogenic response. Compared with WE-AX, AXOS showed a slightly less pronounced effect in the colon as its fermentation was virtually complete in the cecum. Combining WU-AX and AXOS caused a striking synergistic increase in cecal butyrate levels. WU-AX, WE-AX and AXOS together combined a selective bifidogenic effect in the colon with elevated butyrate levels, a reduced pH and suppressed proteolytic metabolites. CONCLUSION: The prebiotic potential and fermentation characteristics of cereal AX depend strongly on their structural properties and joint presence.

Prebiotic and other health-related effects of cereal-derived arabinoxylans, arabinoxylan-oligosaccharides, and xylooligosaccharides.

Arabinoxylans (AX) from cereals are cell wall components that constitute an important part of the dietary fiber intake in humans. Enzymatic hydrolysis of AX yields arabinoxylan-oligosaccharides (AXOS), consisting of arabinoxylooligosaccharides and xylooligosaccharides (XOS). This reaction takes place in the production of AXOS and of cereal-derived food products such as bread and beer, as well as in the colon upon ingestion of AX. This review mainly focuses on the available evidence that AXOS and XOS exert prebiotic effects in the colon of humans and animals through selective stimulation of beneficial intestinal microbiota. In addition, in vitro experiments and in vivo intervention studies on animals or humans are discussed that have investigated potential health-related effects resulting from the dietary intake of AX, AXOS, or XOS.

Safety assessment of a wheat bran extract containing arabinoxylan-oligosaccharides: mutagenicity, clastogenicity, and 90-day rat-feeding studies.

Wheat bran extract (WBE) is a food-grade preparation that is highly enriched in arabinoxylan-oligosaccharides. As part of the safety evaluation of WBE, its genotoxic potential was assessed in a bacterial reverse mutagenicity assay (Ames test) and a chromosome aberration assay on Chinese hamster lung fibroblast cells. These in vitro genotoxicity assays showed no evidence of mutagenic or clastogenic activity with WBE. The safety of WBE was furthermore evaluated in a subchronic toxicity study on rats that were fed a semisynthetic diet (AIN 93G) containing 0.3%, 1.5%, or 7.5% WBE for 13 weeks, corresponding to an average intake of 0.2, 0.9, and 4.4 g/kg body weight (bw) per day, with control groups receiving the unsupplemented AIN 93G, AIN 93G with 7.5% inulin, or AIN 93G with 7.5% wheat bran. Based on this rat-feeding study, the no-observed-adverse-effect level (NOAEL) for WBE was determined as 4.4 g/kg (bw)/d, the highest dose tested.

Tolerance of arabinoxylan-oligosaccharides and their prebiotic activity in healthy subjects: a randomised, placebo-controlled cross-over study.

The tolerance and prebiotic effect following oral intake by healthy human subjects of arabinoxylan-oligosaccharides (AXOS), produced by partial enzymic hydrolysis of the wheat fibre arabinoxlyan, were studied. A total of twenty healthy subjects participated in the present randomised, placebo-controlled cross-over study. They consumed 10 g AXOS or placebo per d each for 3 weeks with a 4-week wash-out period in between. Before and immediately after each intake period, blood samples were taken to measure haematological and clinical chemistry parameters and the subjects completed a questionnaire about gastrointestinal symptoms. Additionally, urine was collected over 48 h for analysis of p-cresol and phenol content by GC-MS, and faeces were collected over 72 h for analysis of microbiota using real-time PCR. Of the subjects, ten also performed a urine and faeces collection 2 weeks after the start of intake (during intervention). A limited number of tested blood parameters were influenced in a statistically significantly way by either AXOS or placebo intake, but these changes remained within the normal range. Blood lipids remained unchanged. AXOS had no statistically significant effect on the range of gastrointestinal symptoms, except for a mild increase in flatulence. Urinary p-cresol excretion, an indicator of protein fermentation, was significantly decreased after 2 weeks of AXOS intake. The levels of bifidobacteria were significantly increased after 2 and 3 weeks of AXOS intake as well as after 3 weeks of placebo. However, the effect of AXOS on bifidobacteria was more pronounced than that of placebo. In conclusion, AXOS are a well-tolerated prebiotic at the dose of 10 g/d. AXOS intake increases faecal bifidobacteria and reduces urinary p-cresol excretion.

Arabinoxylan-oligosaccharides (AXOS) reduce preneoplastic lesions in the colon of rats treated with 1,2-dimethylhydrazine (DMH).

BACKGROUND: Prebiotics are non-digestible compounds that beneficially affect the host by stimulating the growth and/or activity of one or a limited number of resident colonic bacteria in the gut. Reported beneficial effects of prebiotics include reduced gut infections, better absorption of minerals, and notably, antitumorigenic effects. Arabinoxylan (AX)-oligosaccharides (AXOS) have been suggested to exert prebiotic effects in the gut, but their effect on colon carcinogenesis has not been studied so far. AIM OF THE STUDY: To test the effect of AXOS in a rat colon carcinogenesis model. METHODS: We determined the occurrence of two types of preneoplastic lesions [aberrant crypt foci (ACF) and mucin depleted foci (MDF)] in the colon of rats treated with the colon carcinogen 1,2-dimethylhydrazine (DMH) and fed either a control diet or a diet containing AXOS (4.8% w/w) (15 rats in each group). RESULTS: Thirteen weeks after DMH treatment, MDF counts were significantly lower in the entire colon of AXOS fed rats (MDF/colon were 7.5 +/- 0.6 and 5.5 +/- 0.6, in Control and AXOS groups, respectively, means +/- SE, P < 0.05). Although the number of ACF in the entire colon was not significantly different between Control and AXOS fed rats, AXOS fed rats had significantly fewer ACF in the distal part of the colon than Control group rats (ACF/distal colon were 135.5 +/- 15 and 84.4 +/- 11, in Control and AXOS groups, respectively, means +/- SE, P < 0.05). CONCLUSIONS: The present study shows that dietary intake of AXOS by rats reduces the occurrence of two types of preneoplastic lesions, thus suggesting a chemopreventive effect on colon carcinogenesis that should be confirmed in a long-term carcinogenesis experiment.

Comparison of prebiotic effects of arabinoxylan oligosaccharides and inulin in a simulator of the human intestinal microbial ecosystem.

In this study, the prebiotic potential of arabinoxylan oligosaccharides (AXOS) was compared with inulin in two simulators of the human intestinal microbial ecosystem. Microbial breakdown of both oligosaccharides and short-chain fatty acid production was colon compartment specific, with ascending and transverse colon being the predominant site of inulin and AXOS degradation, respectively. Lactate levels (+5.5 mM) increased in the ascending colon during AXOS supplementation, while propionate levels (+5.1 mM) increased in the transverse colon. The concomitant decrease in lactate in the transverse colon suggests that propionate was partially formed over the acrylate pathway. Furthermore, AXOS supplementation strongly decreased butyrate in the ascending colon, this in parallel with a decrease in Roseburia spp. and Bacteroides/Prevotella/Porphyromonas (-1.4 and -2.0 log CFU) levels. Inulin treatment had moderate effects on lactate, propionate and butyrate levels. Denaturing gradient gel electrophoresis analysis revealed that inulin changed microbial metabolism by modulating the microbial community composition. In contrast, AXOS primarily affected microbial metabolism by 'switching on' AXOS-degrading enzymes (xylanase, arabinofuranosidase and xylosidase), without significantly affecting microbial community composition. Our results demonstrate that AXOS has a higher potency than inulin to shift part of the sugar fermentation toward the distal colon parts. Furthermore, due to its stronger propionate-stimulating effect, AXOS is a candidate prebiotic capable of lowering cholesterol and beneficially affecting fat metabolism of the host.

Structurally different wheat-derived arabinoxylooligosaccharides have different prebiotic and fermentation properties in rats.

To evaluate the prebiotic potential and intestinal fermentation products of wheat bran-derived arabinoxylooligosaccharides (AXOS) in relation to their structure, 5 preparations with structurally different AXOS were included ( approximately 4% wt:wt) in rat diets that mimicked the average Western human diet composition. Xylooligosaccharides (XOS), fructooligosaccharides (FOS), and inulin were used as references. The observed effects mainly depended on the average degree of polymerization (avDP) of the AXOS preparations. The AXOS and XOS preparations with a low avDP (

Dose-response effect of arabinoxylooligosaccharides on gastrointestinal motility and on colonic bacterial metabolism in healthy volunteers.

OBJECTIVE: Arabinoxylooligosaccharides (AXOS) are non-digestible in the upper gastrointestinal tract and have been shown to exert prebiotic effects in animals. The aim of this study was to characterize the influence of AXOS with an average degree of polymerization of 15 and an average degree of arabinose substitution of 0.26 (AXOS-15-0.26) on gastrointestinal motility and colonic bacterial metabolism in healthy human volunteers. METHODS: Twelve healthy volunteers received five test meals, containing different amounts of AXOS-15-0.26, with one week intervals between each test meal. Breath tests were used to measure gastric emptying rate, oro-cecal transit time (OCTT) and hydrogen excretion. Colonic bacterial metabolism was estimated using the biomarkers lactose-[(15)N, (15)N']-ureide ((15)N-LU) and p-cresol. RESULTS: Gastric emptying and OCTT were not influenced by addition of varying amounts of AXOS-15-0.26. Administration of 2.2g or 4.9 g AXOS-15-0.26 significantly decreased the urinary (15)N-excretion (respectively p = 0.008 and p = 0.035) as compared to the baseline, whereas fecal (15)N-excretion was significantly increased (respectively p = 0.034 and p = 0.019). This shift from urinary to fecal (15)N-excretion suggests a higher uptake or incorporation by bacteria due to the stimulation of colonic bacterial growth and/or metabolic activity. Furthermore, a significant increase in hydrogen excretion after administration of 2.2g (p = 0.002) and 4.9 g (p = 0.004) AXOS-15-0.26 was observed. No influence on urinary p-cresol excretion was observed. CONCLUSION: These findings suggest that a minimal dose of 2.2g AXOS-15-0.26 favorably modulates the colonic bacterial metabolism in healthy humans. However, long term studies are required to confirm a possible prebiotic effect.

The cyclin-dependent kinase inhibitor Orysa;KRP1 plays an important role in seed development of rice.

Kip-related proteins (KRPs) play a major role in the regulation of the plant cell cycle. We report the identification of five putative rice (Oryza sativa) proteins that share characteristic motifs with previously described plant KRPs. To investigate the function of KRPs in rice development, we generated transgenic plants overexpressing the Orysa;KRP1 gene. Phenotypic analysis revealed that overexpressed KRP1 reduced cell production during leaf development. The reduced cell production in the leaf meristem was partly compensated by an increased cell size, demonstrating the existence of a compensatory mechanism in monocot species by which growth rate is less reduced than cell production, through cell expansion. Furthermore, Orysa;KRP1 overexpression dramatically reduced seed filling. Sectioning through the overexpressed KRP1 seeds showed that KRP overproduction disturbed the production of endosperm cells. The decrease in the number of fully formed seeds was accompanied by a drop in the endoreduplication of endosperm cells, pointing toward a role of KRP1 in connecting endocycle with endosperm development. Also, spatial and temporal transcript detection in developing seeds suggests that Orysa;KRP1 plays an important role in the exit from the mitotic cell cycle during rice grain formation.

The role of ethylene in host-pathogen interactions.

The phytohormone ethylene is a principal modulator in many aspects of plant life, including various mechanisms by which plants react to pathogen attack. Induced ethylene biosynthesis and subsequent intracellular signaling through a single conserved pathway have been well characterized. This leads to a cascade of transcription factors consisting of primary EIN3-like regulators and downstream ERF-like transcription factors. The latter control the expression of various effector genes involved in various aspects of systemic induced defense responses. Moreover, at this level significant cross-talk occurs with other defense response pathways controlled by salicylic acid and jasmonate, eventually resulting in a differentiated disease response.

Antifungal activity of synthetic peptides derived from Impatiens balsamina antimicrobial peptides Ib-AMP1 and Ib-AMP4.

Seeds of Impatiens balsamina contain a set of related antimicrobial peptides (Ib-AMPs). We have produced a synthetic variant of Ib-AMP1, oxidized to the bicyclic native conformation, which was fully active on yeast and fungal strains; and four linear 20-mer Ib-AMP variants, including two all-D forms. We show that the all-D variants are as active on yeast and fungal strains as native peptides. In addition, fungal growth inhibition nor salt-dependency of Ib-AMP4 could be improved by more than two-fold via replacement of amino acid residues by arginine or tryptophan. Native Ib-AMPs showed no hemolytic nor toxic activity up to a concentration of 100 microM. All these data demonstrate the potential of the native Ib-AMPs to combat fungal infections.

Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions.

Basic and applied research involving transgenic plants often requires consistent high-level expression of transgenes. However, high inter-transformant variability of transgene expression caused by various phenomena, including gene silencing, is frequently observed. Here, we show that stable, high-level transgene expression is obtained using Arabidopsis thaliana post-transcriptional gene silencing (PTGS) sgs2 and sgs3 mutants. In populations of first generation (T1) A. thaliana plants transformed with a beta-glucuronidase (GUS) gene (uidA) driven by the 35S cauliflower mosaic virus promoter (p35S), the incidence of highly expressing transformants shifted from 20% in wild type background to 100% in sgs2 and sgs3 backgrounds. Likewise, when sgs2 mutants were transformed with a cyclin-dependent kinase inhibitor 6 gene under control of p35S, all transformants showed a clear phenotype typified by serrated leaves, whereas such phenotype was only observed in about one of five wild type transformants. p35S-driven uidA expression remained high and steady in T2 sgs2 and sgs3 transformants, in marked contrast to the variable expression patterns observed in wild type T2 populations. We further show that T-DNA constructs flanked by matrix attachment regions of the chicken lysozyme gene (chiMARs) cause a boost in GUS activity by fivefold in sgs2 and 12-fold in sgs3 plants, reaching up to 10% of the total soluble proteins, whereas no such boost is observed in the wild type background. MAR-based plant transformation vectors used in a PTGS mutant background might be of high value for efficient high-throughput screening of transgene-based phenotypes as well as for obtaining extremely high transgene expression in plants.

A set of modular plant transformation vectors allowing flexible insertion of up to six expression units.

We have constructed a binary vector for Agrobacterium-mediated plant transformation, which has a multiple cloning site consisting of 13 hexanucleotide restriction sites, 6 octanucleotide restriction sites and 5 homing endonuclease sites. The homing endonuclease sites have the advantages to be extremely rare in natural sequences and to allow unidirectional cloning. We have also constructed a set of auxiliary vectors allowing the assembly of expression cassettes flanked by homing endonuclease sites. The expression cassettes assembled in these auxiliary vectors can be transferred into the binary vector with virtually no risk of cutting the vector within previously introduced sequences. This vector set is ideally suited for the construction of plant transformation vectors containing multiple expression cassettes and/or other elements such as matrix attachment regions. With this modular vector system, six different expression units were constructed in as many auxiliary vectors and assembled together in one plant transformation vector. The transgenic nature of Arabidopsis thaliana plants, transformed with this plant transformation vector, was assessed and the expression of each of the six genes was demonstrated.

Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins.

We developed a method for expression in Arabidopsis of a transgene encoding a cleavable chimeric polyprotein. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), DmAMP1 originating from Dahlia merckii seeds and RsAFP2 originating from Raphanus sativus seeds, which are linked by an intervening sequence ("linker peptide") originating from a natural polyprotein occurring in seed of Impatiens balsamina. The chimeric polyprotein was found to be cleaved in transgenic Arabidopsis plants and the individual AMPs were secreted into the extracellular space. Both AMPs were found to exert antifungal activity in vitro. It is surprising that the amount of AMPs produced in plants transformed with some of the polyprotein transgene constructs was significantly higher compared with the amount in plants transformed with a transgene encoding a single AMP, indicating that the polyprotein expression strategy may be a way to boost expression levels of small proteins.