Publications - Cell and Metabolic Biology

In vivo localization of proteins using fluorescence-based approaches by fusion of the protein of interest (POI) to a fluorescent protein is a cost- and time-effective tool to gain insights into its physiological function in a plant cell. Determining the proper localization, however, requires the co-expression of defined organelle markers (OM). Several marker sets are available but, so far, the procedure requires successful co-transformation of POI and OM into the same cell and/or several cloning steps. We developed a set of vectors containing markers for basic cell organelles that enables the insertion of the gene of interest (GOI) by a single cloning step using the Golden Gate cloning approach and resulting in POI–GFP fusions. The set includes markers for plasma membrane, tonoplast, nucleus, endoplasmic reticulum, Golgi apparatus, peroxisomes, plastids, and mitochondria, all labelled with mCherry. Most of them were derived from well-established marker sets, but those localized in plasma membrane and tonoplast were improved by using different proteins. The final vectors are usable for localization studies in isolated protoplasts and for transient transformation of leaves of Nicotiana benthamiana. Their functionality is demonstrated using two enzymes involved in biosynthesis of jasmonic acid and located in either plastids or peroxisomes.

The leaves of the wild tomato Solanum galapagense harbor type-IV glandular trichomes (GT) that produce high levels of acylsugars (AS), conferring insect resistance. Conversely, domesticated tomatoes (S. lycopersicum) lack type-IV trichomes on the leaves of mature plants, preventing high AS production, thus rendering the plants more vulnerable to insect predation. We hypothesized that cultivated tomatoes engineered to harbor type-IV trichomes on the leaves of adult plants could be insect-resistant. We introgressed the genetic determinants controlling type-IV trichome development from S. galapagense into cv. Micro-Tom (MT) and created a line named “Galapagos-enhanced trichomes” (MT-Get). Mapping-by-sequencing revealed that five chromosomal regions of S. galapagense were present in MT-Get. Further genetic mapping showed that S. galapagense alleles in chromosomes 1, 2, and 3 were sufficient for the presence of type-IV trichomes on adult organs but at lower densities. Metabolic and gene expression analyses demonstrated that type-IV trichome density was not accompanied by the AS production and exudation in MT-Get. Although the plants produce a significant amount of acylsugars, those are still not enough to make them resistant to whiteflies. We demonstrate that type-IV glandular trichome development is insufficient for high AS accumulation. The results from our study provided additional insights into the steps necessary for breeding an insect-resistant tomato.

Abstract: Black pepper (Piper nigrum) is among the world’s most popular spices. Its pungent principle, piperine, has already been identified 200 years ago, yet the biosynthesis of piperine in black pepper remains largely enigmatic. In this report we analyzed the characteristic methylenedioxy bridge formation of the aromatic part of piperine by a combination of RNA-sequencing, functional expression in yeast, and LC-MS based analysis of substrate and product profiles. We identified a single cytochrome P450 transcript, specifically expressed in black pepper immature fruits. The corresponding gene was functionally expressed in yeast (Saccharomyces cerevisiae) and characterized for substrate specificity with a series of putative aromatic precursors with an aromatic vanilloid structure. Methylenedioxy bridge formation was only detected when feruperic acid (5-(4-hydroxy-3-methoxyphenyl)-2,4-pentadienoic acid) was used as a substrate, and the corresponding product was identified as piperic acid. Two alternative precursors, ferulic acid and feruperine, were not accepted. Our data provide experimental evidence that formation of the piperine methylenedioxy bridge takes place in young black pepper fruits after a currently hypothetical chain elongation of ferulic acid and before the formation of the amide bond. The partially characterized enzyme was classified as CYP719A37 and is discussed in terms of specificity, storage, and phylogenetic origin of CYP719 catalyzed reactions in magnoliids and eudicots.

The phenotype of the tomato mutant jasmonate-insensitive1-1 (jai1-1) mutated in the JA-Ile co-receptor COI1 demonstrates JA function in flower development, since it is female-sterile. In addition, jai1-1 exhibits a premature anther dehydration and pollen release, being in contrast to a delayed anther dehiscence in the JA-insensitive Arabidopsis mutant coi1-1. The double mutant jai1-1 Never ripe (jai1-1 Nr), which is in addition insensitive to ethylene (ET), showed a rescue of the jai1-1 phenotype regarding pollen release. This suggests that JA inhibits a premature rise in ET to prevent premature stamen desiccation. To elucidate the interplay of JA and ET in more detail, stamen development in jai1-1 Nr was compared to wild type, jai1-1 and Nr regarding water content, pollen vitality, hormone levels, and accumulation of phenylpropanoids and transcripts encoding known JA- and ET-regulated genes. For the latter, RT-qPCR based on nanofluidic arrays was employed. The data showed that additional prominent phenotypic features of jai1-1, such as diminished water content and pollen vitality, and accumulation of phenylpropanoids were at least partially rescued by the ET-insensitivity. Hormone levels and accumulation of transcripts were not affected. The data revealed that strictly JA-regulated processes cannot be rescued by ET-insensitivity, thereby emphasizing a rather minor role of ET in JA-regulated stamen development.

Jasmonates (JAs) are lipid-derived signals in plant stress responses and development. A crucial step in JA biosynthesis is catalyzed by allene oxide cyclase (AOC). Four genes encoding functional AOCs (AOC1, AOC2, AOC3 and AOC4) have been characterized for Arabidopsis thaliana in terms of organ- and tissue-specific expression, mutant phenotypes, promoter activities and initial in vivo protein interaction studies suggesting functional redundancy and diversification, including first hints at enzyme activity control by protein-protein interaction. Here, these analyses were extended by detailed analysis of recombinant proteins produced in Escherichia coli. Treatment of purified AOC2 with SDS at different temperatures, chemical cross-linking experiments and protein structure analysis by molecular modelling approaches were performed. Several salt bridges between monomers and a hydrophobic core within the AOC2 trimer were identified and functionally proven by site-directed mutagenesis. The data obtained showed that AOC2 acts as a trimer. Finally, AOC activity was determined in heteromers formed by pairwise combinations of the four AOC isoforms. The highest activities were found for heteromers containing AOC4 + AOC1 and AOC4 + AOC2, respectively. All data are in line with an enzyme activity control of all four AOCs by heteromerization, thereby supporting a putative fine-tuning in JA formation by various regulatory principles.

In oilseed rape (Brassica napus), the glucosyltransferase UGT84A9 catalyzes the formation of 1-O-sinapoyl-β-glucose, which feeds as acyl donor into a broad range of accumulating sinapate esters, including the major antinutritive seed component sinapoylcholine (sinapine). Since down-regulation of UGT84A9 was highly efficient in decreasing the sinapate ester content, the genes encoding this enzyme were considered as potential targets for molecular breeding of low sinapine oilseed rape. B. napus harbors two distinguishable sequence types of the UGT84A9 gene designated as UGT84A9-1 and UGT84A9-2. UGT84A9-1 is the predominantly expressed variant, which is significantly up-regulated during the seed filling phase, when sinapate ester biosynthesis exhibits strongest activity. In the allotetraploid genome of B. napus, UGT84A9-1 is represented by two loci, one derived from the Brassica C-genome (UGT84A9a) and one from the Brassica A-genome (UGT84A9b). Likewise, for UGT84A9-2 two loci were identified in B. napus originating from both diploid ancestor genomes (UGT84A9c, Brassica C-genome; UGT84A9d, Brassica A-genome). The distinct UGT84A9 loci were genetically mapped to linkage groups N15 (UGT84A9a), N05 (UGT84A9b), N11 (UGT84A9c) and N01 (UGT84A9d). All four UGT84A9 genomic loci from B. napus display a remarkably low micro-collinearity with the homologous genomic region of Arabidopsis thaliana chromosome III, but exhibit a high density of transposon-derived sequence elements. Expression patterns indicate that the orthologous genes UGT84A9a and UGT84A9b should be considered for mutagenesis inactivation to introduce the low sinapine trait into oilseed rape.

Resveratrol is a phytoalexin produced in various plants like wine, peanut or pine in response to fungal infection or UV irradiation, but it is absent in members of the Brassicaceae. Moreover, resveratrol and its glucoside (piceid) are considered to have beneficial effects on human health, known to reduce heart disease, arteriosclerosis and cancer mortality. Therefore, the introduction of the gene encoding stilbene synthase for resveratrol production in rapeseed is a tempting approach to improve the quality of rapeseed products. The stilbene synthase gene isolated from grapevine (Vitis vinifera L.) was cloned under control of the seed-specific napin promotor and introduced into rapeseed (Brassica napus L.) by Agrobacterium-mediated co-transformation together with a ds-RNA-interference construct deduced from the sequence of the key enzyme for sinapate ester biosynthesis, UDP-glucose:sinapate glucosyltransferase (BnSGT1), assuming that the suppression of the sinapate ester biosynthesis may increase the resveratrol production in seeds through the increased availability of the precursor 4-coumarate. Resveratrol glucoside (piceid) was produced at levels up to 361 μg/g in the seeds of the primary transformants. This value exceeded by far piceid amounts reported from B. napus expressing VST1 in the wild type sinapine background. There was no significant difference in other important agronomic traits, like oil, protein, fatty acid and glucosinolate content in comparison to the control plants. In the third seed generation, up to 616 μg/g piceid was found in the seeds of a homozygous T3-plant with a single transgene copy integrated. The sinapate ester content in this homozygous T3-plant was reduced from 7.43 to 2.40 mg/g. These results demonstrate how the creation of a novel metabolic sink could divert the synthesis towards the production of piceid rather than sinapate ester, thereby increasing the value of oilseed products.