Rita Zrenner

UV-B-Induced Secondary Plant Metabolites - Potential Benefits for Plant and Human Health

Epidemiological studies have revealed an inverse association between the consumption of fruit, vegetables, and herbs and the risk of both cancer and cardiovascular disease. This protective effect is mostly due to secondary metabolites present in plant tissues. During the last decade, it has become increasingly clear that UV-B radiation is an important regulator of plant secondary metabolism. Low, ecologically-relevant UV-B levels trigger distinct changes in the accumulation of, among others, phenolic compounds, carotenoids and glucosinolates. Fundamental understanding of plant UV-B perception and responses opens up new opportunities for crop manipulation. Thus, targeted low dosage UV-B radiation treatments as emerging technology may be used to generate fruit, vegetables, and herbs enriched with secondary plant metabolites for either fresh consumption or as a source for functional foods and nutraceuticals, resulting in increased ingestion of these health-promoting substances. The UV-B induced accumulation of secondary plant metabolites is likely to have evolved as a plant defense response against harmful UV-B radiation. However, UV-B induced secondary metabolites also alter other trophic interactions, for example by altering plant herbivore resistance. Thus, UV-B driven metabolic changes in the plants secondary metabolism have benefits for both ends of the bio-based food chain, i.e., for plants themselves as well as for humans.

UV-B irradiation changes specifically the secondary metabolite profile in broccoli sprouts: induced signaling overlaps with defense response to biotic stressors.

Only a few environmental factors have such a pronounced effect on plant growth and development as ultraviolet light (UV). Concerns have arisen due to increased UV-B radiation reaching the Earth’s surface as a result of stratospheric ozone depletion. Ecologically relevant low to moderate UV-B doses (0.3–1 kJ m–2 d–1) were applied to sprouts of the important vegetable crop Brassica oleracea var. italica (broccoli), and eco-physiological responses such as accumulation of non-volatile secondary metabolites were related to transcriptional responses with Agilent One-Color Gene Expression Microarray analysis using the 2×204 k format Brassica microarray. UV-B radiation effects have usually been linked to increases in phenolic compounds. As expected, the flavonoids kaempferol and quercetin accumulated in broccoli sprouts (the aerial part of the seedlings) 24 h after UV-B treatment. A new finding is the specific UV-B-mediated induction of glucosinolates (GS), especially of 4-methylsulfinylbutyl GS and 4-methoxy-indol-3-ylmethyl GS, while carotenoids and Chl levels remained unaffected. Accumulation of defensive GS metabolites was accompanied by increased expression of genes associated with salicylate and jasmonic acid signaling defense pathways and up-regulation of genes responsive to fungal and bacterial pathogens. Concomitantly, plant pre-exposure to moderate UV-B doses had negative effects on the performance of the caterpillar Pieris brassicae (L.) and on the population growth of the aphid Myzus persicae (Sulzer). Moreover, insect-specific induction of GS in broccoli sprouts was affected by UV-B pre-treatment.

Induced Production of 1-Methoxy-indol-3-ylmethyl Glucosinolate by Jasmonic Acid and Methyl Jasmonate in Sprouts and Leaves of Pak Choi (Brassica rapa ssp. chinensis)

Pak choi plants (Brassica rapa ssp. chinensis) were treated with different signaling molecules methyl jasmonate, jasmonic acid, linolenic acid, and methyl salicylate and were analyzed for specific changes in their glucosinolate profile. Glucosinolate levels were quantified using HPLC-DAD-UV, with focus on induction of indole glucosinolates and special emphasis on 1-methoxy-indol-3-ylmethyl glucosinolate. Furthermore, the effects of the different signaling molecules on indole glucosinolate accumulation were analyzed on the level of gene expression using semi-quantitative realtime RT-PCR of selected genes. The treatments with signaling molecules were performed on sprouts and mature leaves to determine ontogenetic differences in glucosinolate accumulation and related gene expression. The highest increase of indole glucosinolate levels, with considerable enhancement of the 1-methoxy-indol-3-ylmethyl glucosinolate content, was achieved with treatments of sprouts and mature leaves with methyl jasmonate and jasmonic acid. This increase was accompanied by increased expression of genes putatively involved in the indole glucosinolate biosynthetic pathway. The high levels of indole glucosinolates enabled the plant to preferentially produce the respective breakdown products after tissue damage. Thus, pak choi plants treated with methyl jasmonate or jasmonic acid, are a valuable tool to analyze the specific protection functions of 1-methoxy-indole-3-carbinole in the plants defense strategy in the future.

Genotypic Variation of the Glucosinolate Profile in Pak Choi (Brassica rapa ssp. chinensis)

Thirteen different pak choi (Brassica rapa ssp. chinensis) cultivars were characterized regarding their glucosinolate profile analyzed by HPLC-DAD-MS. The identified glucosinolates were subjected to principal component analysis, and three distinct groups of pak choi sprouts were identified. Group differences were marked mainly by variations in the aliphatic glucosinolate profile such as differing levels of 3-butenyl glucosinolate and 2-hydroxy-3-butenyl glucosinolate as well as by their varying proportional ratios. In addition, the three groups of pak choi sprouts varied by the presence or absence of 2-hydroxy-4-pentenyl glucosinolate and in level and composition of butyl glucosinolates. This classification is reflected by relative mRNA expression level of 2-oxoacid-dependent dioxygenase. As in sprouts, the major glucosinolates in mature leaves were found to be the aliphatic glucosinolates. However, unlike in sprouts, an additional aliphatic glucosinolate, 5-methylsulfinylpentyl glucosinolate, was detected as characteristic ontogenetic variation in mature leaves in 12 of the 13 pak choi cultivars analyzed.

Vegetable Grafting: The Implications of a Growing Agronomic Imperative for Vegetable Fruit Quality and Nutritive Value

Grafting has become an imperative for intensive vegetable production since chlorofluorocarbon-based soil fumigants were banned from use on grounds of environmental protection. Compelled by this development, research into rootstock–scion interaction has broadened the potential applications of grafting in the vegetable industry beyond aspects of soil phytopathology. Grafting has been increasingly tapped for cultivation under adverse environs posing abiotic and biotic stresses to vegetable crops, thus enabling expansion of commercial production onto otherwise under-exploited land. Vigorous rootstocks have been employed not only in the open field but also under protected cultivation where increase in productivity improves distribution of infrastructural and energy costs. Applications of grafting have expanded mainly in two families: the Cucurbitaceae and the Solanaceae, both of which comprise major vegetable crops. As the main drives behind the expansion of vegetable grafting have been the resistance to soilborne pathogens, tolerance to abiotic stresses and increase in yields, rootstock selection and breeding have accordingly conformed to the prevailing demand for improving productivity, arguably at the expense of fruit quality. It is, however, compelling to assess the qualitative implications of this growing agronomic practice for human nutrition. Problems of impaired vegetable fruit quality have not infrequently been associated with the practice of grafting. Accordingly, the aim of the current review is to reassess how the practice of grafting and the prevalence of particular types of commercial rootstocks influence vegetable fruit quality and, partly, storability. Physical, sensorial and bioactive aspects of quality are examined with respect to grafting for watermelon, melon, cucumber, tomato, eggplant, and pepper. The physiological mechanisms at play which mediate rootstock effects on scion performance are discussed in interpreting the implications of grafting for the configuration of vegetable fruit physicochemical quality and nutritive value.

Influence of Light and Temperature on Gene Expression Leading to Accumulation of Specific Flavonol Glycosides and Hydroxycinnamic Acid Derivatives in Kale (Brassica oleracea var. sabellica)

Light intensity and temperature are very important signals for the regulation of plant growth and development. Plants subjected to less favorable light or temperature conditions often respond with accumulation of secondary metabolites. Some of these metabolites have been identified as bioactive compounds, considered to exert positive effects on human health when consumed regularly. In order to test a typical range of growth parameters for the winter crop Brassica oleracea var. sabellica, plants were grown either at 400 μmol m−2 s−1 or 100 μmol m−2 s−1 at 10°C, or at 400 μmol m−2 s−1 with 5 or 15°C. The higher light intensity overall increased flavonol content of leaves, favoring the main quercetin glycosides, a caffeic acid monoacylated kaempferol triglycoside, and disinapoyl-gentiobiose. The higher temperature mainly increased the hydroxycinnamic acid derivative disinapoyl-gentiobiose, while at lower temperature synthesis is in favor of very complex sinapic acid acylated flavonol tetraglycosides such as kaempferol-3-O-sinapoyl-sophoroside-7-O-diglucoside. A global analysis of light and temperature dependent alterations of gene expression in B. oleracea var. sabellica leaves was performed with the most comprehensive Brassica microarray. When compared to the light experiment much less genes were differentially expressed in kale leaves grown at 5 or 15°C. A structured evaluation of differentially expressed genes revealed the expected enrichment in the functional categories of e.g. protein degradation at different light intensities or phytohormone metabolism at different temperature. Genes of the secondary metabolism namely phenylpropanoids are significantly enriched with both treatments. Thus, the genome of B. oleracea was screened for predicted genes putatively involved in the biosynthesis of flavonoids and hydroxycinnamic acid derivatives. All identified B. oleracea genes were analyzed for their most specific 60-mer oligonucleotides present on the 2 × 105 K format Brassica microarray. Expression differences were correlated to the structure-dependent response of flavonoid glycosides and hydroxycinnamic acid derivatives to alterations in either light or temperature. The altered metabolite accumulation was mainly reflected on gene expression level of core biosynthetic pathway genes and gave further hints to an isoform specific functional specialization.

Low and moderate photosynthetically active radiation affects the flavonol glycosides and hydroxycinnamic acid derivatives in kale (Brassica oleracea var. sabellica) dependent on two low temperatures.

Kale (Brassica oleracea var. sabellica) contains a large number of naturally occurring structurally different non-acylated and acylated flavonol glycosides as well as hydroxycinnamic acid derivatives. The objective of this study was to determine the effect of low and moderate photosynthetic active radiation (PAR) and how these levels interact with low temperature in these phenolic compounds. Juvenile kale plants were treated with PAR levels from 200 to 800 μmol m(-2) s(-1) at 5 and 10 °C under defined conditions in climate chambers. Of the investigated 20 compounds, 11 and 17 compounds were influenced by PAR and temperature, respectively. In addition, an interaction between PAR and temperature was found for eight compounds. The response of the phenolic compounds to PAR was structure-dependent. While quercetin triglycosides increased with higher PAR at 5 and 10 °C, the kaempferol triglycosides exhibited the highest concentrations at 400 μmol m(-2) s(-1). In contrast, kaempferol diglycosides exhibited the highest concentrations at increased PAR levels of 600 and 800 μmol m(-2) s(-1) at 10 °C. However, key genes of flavonol biosynthesis were influenced by temperature but remained unaffected by PAR. Furthermore, there was no interaction between the PAR level and the low temperature in the response of hydroxycinnamic acid derivatives in kale with the exception of caffeoylquinic acid, which decreased with higher PAR levels of 600 and 800 μmol m(-2) s(-1) and at a lower temperature. In conclusion, PAR and its interaction with temperature could be a suitable tool for modifying the profile of phenolic compounds.

UV-B Elicitation of Secondary Plant Metabolites

Epidemiological studies have revealed an inverse association between a high consumption of vegetables and a lower risk of both cancer and cardiovascular diseases. This protective effect is mostly due to secondary plant metabolites present in plant tissues. In this context, it has become increasingly clear during the last decade that UV-B radiation is an important regulator of plant secondary metabolism. Recent studies have highlighted the regulatory properties of low, ecologically relevant UV-B levels contrary to previous studies in which UV-B radiation was exclusively regarded as a stress factor. Low-dosage UV-B applications trigger distinct changes in the plant’s secondary metabolism resulting in an accumulation of phenolic compounds such as flavonoids and glucosinolates.

Metabolic Engineering of Aliphatic Glucosinolates in Hairy Root Cultures of Arabidopsis thaliana

Two cytochrome P450 proteins, CYP79F1 and CYP79F2, are involved in the biosynthesis of aliphatic glucosinolates (GSs) in brassicaceous species. In order to increase aliphatic GS levels in Arabidopsis thaliana hairy root cultures (HRCs) with the aim of biomanufacturing GSs, CYP79F1 and CYP79F2 from either Arabidopsis thaliana or Brassica rapa were first constitutively expressed in stably transformed Arabidopsis plants, and then HRC was induced by additional transformation with Agrobacterium rhizogenes. Gene expression levels and GS profiles were analyzed in HRC in order to validate the impact on aliphatic levels. To estimate the potency of gene transformation on GS content in HRC, GS levels were compared to levels in roots and leaves of transformed plants. According to the data obtained, short-chain aliphatic GSs in plants were raised in three generations after transformation, indicating that expression of transgenes was stable. After HRC induction, transcript levels of CYP79F1 and CYP79F2 rose and short-chain aliphatic GS levels increased by as much as 7-fold compared to the respective HRC wild-type control. However, when compared to levels present in leaves and roots of plants before HRC induction, aliphatic GS levels in HRC remained very low. In addition, expression of CYP79F1 and CYP79F2 also enhanced the content of indole GSs, mainly indol-3-ylmethyl GS and 4-hydroxy-indol-3-ylmethyl GS in HRC. The results presented provide basic information about GS biosynthesis in HRC of Arabidopsis thaliana compared to intact plants, which will help scientists to further optimize GS composition in HRC in the future.

The Rhizoctonia solani AG1-IB (isolate 7/3/14) transcriptome during interaction with the host plant lettuce (Lactuca sativa L.)

The necrotrophic pathogen Rhizoctonia solani is one of the most economically important soil-borne pathogens of crop plants. Isolates of R. solani AG1-IB are the major pathogens responsible for bottom-rot of lettuce (Lactuca sativa L.) and are also responsible for diseases in other plant species. Currently, there is lack of information regarding the molecular responses in R. solani during the pathogenic interaction between the necrotrophic soil-borne pathogen and its host plant. The genome of R. solani AG1-IB (isolate 7/3/14) was recently established to obtain insights into its putative pathogenicity determinants. In this study, the transcriptional activity of R. solani AG1-IB was followed during the course of its pathogenic interaction with the host plant lettuce under controlled conditions. Based on visual observations, three distinct pathogen-host interaction zones on lettuce leaves were defined which covered different phases of disease progression on tissue inoculated with the AG1-IB (isolate 7/3/14). The zones were defined as: Zone 1—symptomless, Zone 2—light brown discoloration, and Zone 3—dark brown, necrotic lesions. Differences in R. solani hyphae structure in these three zones were investigated by microscopic observation. Transcriptional activity within these three interaction zones was used to represent the course of R. solani disease progression applying high-throughput RNA sequencing (RNA-Seq) analysis of samples collected from each Zone. The resulting three transcriptome data sets were analyzed for their highest expressed genes and for differentially transcribed genes between the respective interaction zones. Among the highest expressed genes was a group of not previously described genes which were transcribed exclusively during early stages of interaction, in Zones 1 and 2. Previously described importance of up-regulation in R. solani agglutinin genes during disease progression could be further confirmed; here, the corresponding genes exhibited extremely high transcription levels. Most differentially higher expressed transcripts were found within Zone 2. In Zone 3, the zone with the strongest degree of interaction, gene transcripts indicative of apoptotic activity were highly abundant. The transcriptome data presented in this work support previous models of the disease and interaction cycle of R. solani and lettuce and may influence effective techniques for control of this pathogen.