Sven Bode Andersen

Molecular activities, biosynthesis and evolution of triterpenoid saponins

Saponins are bioactive compounds generally considered to be produced by plants to counteract pathogens and herbivores. Besides their role in plant defense, saponins are of growing interest for drug research as they are active constituents of several folk medicines and provide valuable pharmacological properties. Accordingly, much effort has been put into unraveling the modes of action of saponins, as well as in exploration of their potential for industrial processes and pharmacology. However, the exploitation of saponins for bioengineering crop plants with improved resistances against pests as well as circumvention of laborious and uneconomical extraction procedures for industrial production from plants is hampered by the lack of knowledge and availability of genes in saponin biosynthesis. Although the ability to produce saponins is rather widespread among plants, a complete synthetic pathway has not been elucidated in any single species. Current conceptions consider saponins to be derived from intermediates of the phytosterol pathway, and predominantly enzymes belonging to the multigene families of oxidosqualene cyclases (OSCs), cytochromes P450 (P450s) and family 1 UDP-glycosyltransferases (UGTs) are thought to be involved in their biosynthesis. Formation of unique structural features involves additional biosynthetical enzymes of diverse phylogenetic background. As an example of this, a serine carboxypeptidase-like acyltransferase (SCPL) was recently found to be involved in synthesis of triterpenoid saponins in oats. However, the total number of identified genes in saponin biosynthesis remains low as the complexity and diversity of these multigene families impede gene discovery based on sequence analysis and phylogeny. This review summarizes current knowledge of triterpenoid saponin biosynthesis in plants, molecular activities, evolutionary aspects and perspectives for further gene discovery.

Evolutionary Agroecology: the potential for cooperative, high density, weed-suppressing cereals

Evolutionary theory can be applied to improve agricultural yields and/or sustainability, an approach we call Evolutionary Agroecology. The basic idea is that plant breeding is unlikely to improve attributes already favored by millions of years of natural selection, whereas there may be unutilized potential in selecting for attributes that increase total crop yield but reduce plants’ individual fitness. In other words, plant breeding should be based on group selection. We explore this approach in relation to crop‐weed competition, and argue that it should be possible to develop high density cereals that can utilize their initial size advantage over weeds to suppress them much better than under current practices, thus reducing or eliminating the need for chemical or mechanical weed control. We emphasize the role of density in applying group selection to crops: it is competition among individuals that generates the ‘Tragedy of the Commons’, providing opportunities to improve plant production by selecting for attributes that natural selection would not favor. When there is competition for light, natural selection of individuals favors a defensive strategy of ‘shade avoidance’, but a collective, offensive ‘shading’ strategy could increase weed suppression and yield in the high density, high uniformity cropping systems we envision.

Identification of Defense Compounds in Barbarea vulgaris against the Herbivore Phyllotreta nemorum by an Ecometabolomic Approach

Winter cress (Barbarea vulgaris) is resistant to a range of insect species. Some B. vulgaris genotypes are resistant, whereas others are susceptible, to herbivory by flea beetle larvae (Phyllotreta nemorum). Metabolites involved in resistance to herbivory by flea beetles were identified using an ecometabolomic approach. An F2 population representing the whole range from full susceptibility to full resistance to flea beetle larvae was generated by a cross between a susceptible and a resistant B. vulgaris plant. This F2 offspring was evaluated with a bioassay measuring the ability of susceptible flea beetle larvae to survive on each plant. Metabolites that correlated negatively with larvae survival were identified through correlation, cluster, and principal component analyses. Two main clusters of metabolites that correlate negatively with larvae survival were identified. Principal component analysis grouped resistant and susceptible plants as well as correlated metabolites. Known saponins, such as hederagenin cellobioside and oleanolic acid cellobioside, as well as two other saponins correlated significantly with plant resistance. This study shows the potential of metabolomics to identify bioactive compounds involved in plant defense.

A genetic linkage map of Spinacia oleracea and localization of a sex determination locus

A genetic map of Spinach (Spinacia oleracea) was constructed in a classical back cross population using 101 AFLP and 9 microsatellite markers. The map was divided into seven linkage groups with a total length of 585 cM and an average distance between the markers of 5.18 cM. The linkage map was constructed with LOD 3.5, but was quite stable with seven linkage groups remaining until LOD 7.0. Gender segregated 1 male to 1 female in the mapping population and was mapped to a small area of one linkage group with a distance of 1.9 cM to a microsatellite marker termed SO4. This small chromosomal region co-segregating with sex determination in the species is in contrast to previous reports on a heterologous XY chromosome system in spinach. Microsatellite markers used as anchors in the map construction were isolated from sequences of known nuclear encoded genes in spinach. This enabled simultaneous positioning on the map of these genes: Rubisco activase (Rca), Photosytem 1 subunit V (PsaG), Protein Kinase (Pk), Nitrate reductase (Nir), ferrodoxin:thioredoxin reductase (Ftr), Ribosomal protein L1 (Rps22), Choline monooxygenase (Cmo), Pseudogene for BZIP protein (Bzip), Glycerol-3-phosphate acyltransferase (Act1) and stromal ascorbate peroxidase, thylakoid-bound ascorbate peroxidase (Apx2). Spinach has a small genome, which makes it suitable for basic genomic studies and many physiologically important genes have been cloned from the species. The present map anchored with user friendly microsatellite markers will be useful for future studies of physiology and genetics of the species as well as studies of the nature of gender determination.

A comparative study of the allergenic potency of wild-type and glyphosate-tolerant gene-modified soybean cultivars.

A large proportion of soybean cultivars grown in the USA are now genetically modified varieties and concern has been raised about the safety of these products for consumers. A study of the impact on allergenic potency in soybeans, comparable except for the newly introduced gene (CP4 EPSPS), was performed using soybean‐sensitized patients. The allergenicity of 18 different (10 GM and 8 WT) soybean extracts was examined blindly by the following three methods: A) Sera from patients with specific IgE against soybean were used to determine concentrations inducing 50% RAST inhibition; B) Histamine release induced by the extracts was examined using blood from sensitized patients; C) SPT was performed on sensitized patients with all 18 extracts. All three methods showed variations in the allergenic potency between the individual extracts but allergenic potential was not affected by presence of the transgene. By using standard in vitro methods and SPT for determination of allergenicity we were not able to detect any significant difference in the allergenic potency between GM and WT soybeans.

Barbarea vulgaris linkage map and quantitative trait loci for saponins, glucosinolates, hairiness and resistance to the herbivore Phyllotreta nemorum

Combined genomics and metabolomics approaches were used to unravel molecular mechanisms behind interactions between winter cress (Barbarea vulgaris) and flea beetle (Phyllotreta nemorum). B. vulgaris comprises two morphologically, biochemically and cytologically deviating types, which differ in flea beetle resistance, saponin and glucosinolate profiles, as well as leaf pubescence. An F2 population generated from a cross between the two B. vulgaris types was used to construct a B. vulgaris genetic map based on 100 AFLP and 31 microsatellite markers. The map was divided into eight linkage groups. QTL (quantitative trait loci) analysis revealed a total of 15 QTL affecting eight traits, including nine QTL for four saponins, two QTL for two glucosinolates, two QTL for hairiness, and two QTL for flea beetle resistance. The two QTL for resistance towards flea beetles in B. vulgaris co-localized with QTL for the four saponins associated with resistance. Furthermore, global QTL analysis of B. vulgaris metabolites identified QTL for a number of flavonoid glycosides and additional saponins from both resistant and susceptible types. The transcriptome of the resistant B. vulgaris type was sequenced by pyrosequencing, and sequences containing microsatellites were identified. Microsatellite types in B. vulgaris were similar to Arabidopsis thaliana but different from Oryza sativa. Comparative analysis between B. vulgaris and A. thaliana revealed a remarkable degree of synteny between a large part of linkage groups 1 and 4 of B. vulgaris harboring the two QTL for flea beetle resistance and Arabidopsis chromosomes 3 and 1. Gene candidates that may underlie QTL for resistance and saponin biosynthesis are discussed.

Effects of donor plant temperature, photoperiod, and age on anther culture response of Capsicum annuum L.

SummaryEffects of temperature and photoperiod for donor plant growth on embryo formation in Capsicum annuum anther culture were investigated. Donor plants were grown in glasshouses at minimum temperatures between 16 and 30° C and at photoperiods between 11 and 19 h. Anthers were collected from individual plants over five to nine week periods to test the significance of donor plant age. Embryos were obtained at all temperature regimes with a calculated optimum temperature of 26.4° C. Embryo formation was unaffected by the photoperiods tested. Embryo formation varied among successive samplings. However, a significant decline in anther culture response with increasing donor plant age was observed in all three experiments.

Wheat cultivars selected for high Fv /Fm under heat stress maintain high photosynthesis, total chlorophyll, stomatal conductance, transpiration and dry matter.

The chlorophyll fluorescence parameter Fv /Fm reflects the maximum quantum efficiency of photosystem II (PSII) photochemistry and has been widely used for early stress detection in plants. Previously, we have used a three-tiered approach of phenotyping by Fv /Fm to identify naturally existing genetic variation for tolerance to severe heat stress (3 days at 40°C in controlled conditions) in wheat (Triticum aestivum L.). Here we investigated the performance of the previously selected cultivars (high and low group based on Fv /Fm value) in terms of growth and photosynthetic traits under moderate heat stress (1 week at 36/30°C day/night temperature in greenhouse) closer to natural heat waves in North-Western Europe. Dry matter accumulation after 7 days of heat stress was positively correlated to Fv /Fm . The high Fv /Fm group maintained significantly higher total chlorophyll and net photosynthetic rate (PN ) than the low group, accompanied by higher stomatal conductance (gs ), transpiration rate (E) and evaporative cooling of the leaf (ΔT). The difference in PN between the groups was not caused by differences in PSII capacity or gs as the variation in Fv /Fm and intracellular CO2 (Ci ) was non-significant under the given heat stress. This study validated that our three-tiered approach of phenotyping by Fv /Fm performed under increasing severity of heat was successful in identifying wheat cultivars differing in photosynthesis under moderate and agronomically more relevant heat stress. The identified cultivars may serve as a valuable resource for further studies to understand the physiological mechanisms underlying the genetic variability in heat sensitivity of photosynthesis.

Phenotyping of wheat cultivars for heat tolerance using chlorophyll a fluorescence

In view of the global climate change, heat stress is an increasing constraint for the productivity of wheat (Triticum aestivum L.). Our aim was to identify contrasting cultivars in terms of heat tolerance by mass screening of 1274 wheat cultivars of diverse origin, based on a physiological trait, the maximum quantum efficiency of PSII (Fv/Fm). A chlorophyll fluorescence protocol was standardised and used for repeated screening with increased selection pressure with a view to identifying a set of cultivars extreme for the trait. An initial mass screening of 1274 wheat cultivars with a milder heat stress of 38°C in 300µmolm-2s-1 for 2h with preheating at 33-35°C for 19h in 7-14µmolm-2s-1 light showed a genetic determination of 8.5±2.7%. A heat treatment of 40°C in 300µmolm-2s-1 for 72h in the second screening with 138 selected cultivars resulted in larger differentiation of cultivars with an increased genetic component (15.4±3.6%), which was further increased to 27.9±6.8% in the third screening with 41 contrasting cultivars. This contrasting set of cultivars was then used to compare the ability of chlorophyll fluorescence parameters to detect genetic difference in heat tolerance. The identification of a set of wheat cultivars contrasting for their inherent photochemical efficiency may aid future studies to understand the genetic and physiological nature of heat stress tolerance in order to dissect quantitative traits into simpler genetic factors.

Morphological and molecular diversity of Nordic oat through one hundred years of breeding

SummaryGenetic diversity in microsatellites and development of agronomical characters in Nordic oat cultivars (Avena sativa) from the 20th century, ranging from landraces to new cultivars, were studied. A clear development in agronomical characters has taken place in this period: Straw length has been reduced, harvest index has increased and heading date has declined. The persistent oat breeding effort in the northern part of the region was indicated by the data, since cultivars from this region showed higher harvest index. Also adaptation to shorter summers was apparent in cultivars from the most northern part of the area. When comparing cultivars released after 1940 to the landraces, the loss of diversity revealed for the agronomical characters was also indicated by the molecular data. This indicates that a more general loss of diversity has taken place in the period, possibly due to random factors during the breeding process (bottleneck effect). The reduction in diversity revealed by recent cultivars at an agronomical as well as a molecular level emphasises the importance of implementing a conservation strategy for older material in order to secure genetic diversity for future oat breeding efforts.