Evo-devo of flowers and flowering genes: Salvia as a case study

The Plantae kingdom and its autotrophic representatives shape the surface of planet Earth and especially flowers contribute to a great amount to the astonishing beauty of nature. Naturally, flowers are not primarily intended to please humans, but they represent the sexual reproductive organs of the angiosperms, the flowering plants. The ingenious interaction of flowers with pollinators is an example for co-evolution and results in fine-scaled adaptations and complex floral shapes and structures. The unique staminal lever mechanism that is limited to the species rich genus Salvia from the Lamiaceae family is an extraordinary example for plant-pollinator interaction. The species of genus Salvia possess zygomorphic flowers, which have an amazing variety of petals and stamens, resulting in melitto- and ornithophily and worldwide distribution. In order to conduct a robust investigation on the evo-devo of Salvia flowers, the aim of the study was to take as holistic a view as possible on the genus Salvia by interweaving morphological, molecular and developmental approaches and put this against the background of speciation. Flowers of 24 Salvia species from different parts of the world were investigated with regard to their floral geometry. Measurements of corolla and reproductive traits were analyzed by principal component analysis, which unveiled the flower size (represented by 79,21% of data in PCA) and bilabiate or tuberous floral shape (represented by 11,34% of data in PCA) as dominating floral features. In this context, the greater floral variety of New World Salvia species (concerning the dimensions size and shape) could be demonstrated, a result that is congruent with the elevated species number of genus Salvia on these continents. Finally, the statistical evaluation of floral traits appears to be a suitable tool to morphologically classify Salvia species. For characteristic genes from the ABCDE model of flowering orthologues were identified for the genus Salvia and used for gene expression experiments that were conducted in two distant (concerning origin, floral habitus or pollinator) related Salvia species, S. pratensis and S. elegans, and here for closed flower buds and fully opened inflorescences, respectively. Key finding in this experiment is a general gene expression pattern that for the most part corresponds with the ABCDE model demonstrating the models conservation for the biggest genus of the Lamiaceae. Additionally, most of the evaluated flowering genes have been shown to be higher expressed in developing tissue, compared to opened flower tissue. B-class genes display huge elevation in expression in Salvia pratensis and Salvia elegans in corolla and stamen tissue, the organs that are of crucial importance for attracting pollinators and the way of reproductive isolation in Salvia. The two B-class genes GLOBOSA and DEFICIENS have been used for an in-depth Bayesian based phylogenetic inference including more than 30 different species, which is the first examination of a floral trait-related marker for the genus Salvia and therefore directly linked with speciation in this genus. Different levels of gene duplication were unraveled, with a duplication event of the GLOBOSA gene that is limited to New World Salvia species, whereas the DEFICIENS gene displays a clear duplication that is evident for the whole genus Salvia. A model based on the multiplication of transcription factor interactions of flowering genes has been elaborated, hypothesizing that this increase of interactions synchronizes with the species radiation of genus Salvia in the New World and thus might one of the driving forces for this radiation. Significant posterior probability (> 95%) values underline the results and also proves the suitability for phylogenetics using these genes in the genus Salvia, especially for the Eurasian Salvia species. Plotting the amino acid sequences of B-class genes in the wider context of ABCDE genes including sequences from Arabidopsis, lead to a strongly supported ABCDE cluster, that shows a remarkable split into a B-class clade and an ACDE-class clade. This split was interpreted as an early divergence of division of responsibility into plant-based intrinsic factors (ACDE-class), like inward seed growth, and extrinsic factors (B-class), like outward interaction with pollinators, in Salvia flowers or maybe for flowers in general. Taken together, the evo-devo approaches conducted in this thesis and applied to the species rich genus Salvia give an insight on the driving forces behind the speciation process

Tracing tiger nut (C. esculentus L.): functional food from the colossal Cyperus genus

Tiger nuts (also called earth almonds or chufa) are the stolons of the sedge Cyperus esculentus L. and have been tradition- ally used as food or as an additive to drinks such as horchata de chufa. Recently, chufa experienced a hype as functional food because of its richness in dietary fiber and as a source of unsaturated fatty acids. Its close relative, nutgrass (Cyperus rotundus L.) has similarly long history of human use, but rather for medical purposes, due to a high content of secondary compounds. Both species are invasive weeds responsible for high yield losses in crop plants, especially in Southeast Asia, Africa, and subtropical America. They are not easily discerned, occur together, and are both traded under similar or identi- cal vernacular names. For the purpose of consumer protection, it is important to discriminate them in commercial samples, which is not trivial by morphological markers and often impossible since they are mostly used in highly processed form. Therefore, a molecular authentication assay was developed using taxonomically authenticated plants as reference material. The plastidic marker psbA-trnH igs (intergenic spacer) and the nuclear marker Internally Transcribed Spacer were used for barcoding. To develop a sequencing-free fingerprinting assay, a single-nucleotide polymorphism in the ITS2 region could be employed to discriminate C. esculentus from its possible adulterant C. rotundus by a tailored Amplificatory Refractory Mutation System PCR. This approach was successful both, in reference to plants, as well as in highly processed commercial products such as tiger nut flour. We show that by a single PCR we can detect surrogation by nutgrass down to a threshold of 10% in tiger nuts as contribution to safeguard product authenticity against possible adulterations in a growing global marke

Goji Who? Morphological and DNA Based Authentication of a “Superfood”

“Goji” (Lycium barbarum and Lycium chinense) is a generic name for medical plants with a long historical background in the traditional Chinese medicine. With the emerging trend of “Superfoods” several years ago, Goji berries soon became an established product in European countries and not only are the most popular product of traditional Chinese medicine outside of China but to this day one of the symbols of the entire “Superfood” trend. However, since Goji is an umbrella term for different plant species that are closely related, mislabeling and adulterations (unconsciously or purposely) are possible. We carefully verified the identity of Goji reference plant material based on morphological traits, mainly floral structures of several inflorescences of each individual, in order to create a robust background for the downstream applications that were used on those reference plants and additionally on commercial Goji products. We report morphological and molecular based strategies for the differentiation of Lycium barbarum and Lycium chinense. The two different Goji species vary significantly in seed size, with an almost double average seed area in Lycium chinense compared to Lycium barbarum. Differences could be traced on the molecular level as well; using the psbA-trnH barcoding marker, we detected a single nucleotide substitution that was used to develop an easy one-step differentiation tool based on ARMS (amplification refractory mutation system). Two diagnostic primers used in distinct multiplex PCRs yield a second diagnostic band in a subsequent gel electrophoresis for Lycium barbarum or Lycium chinense, respectively. Our ARMS approach is a strong but simple tool to trace either of the two different Goji species. Both the morphological and the molecular analysis showed that all of the tested commercial Goji products contained fruits of the species Lycium barbarum var. barbarum, leading to the assumption that consumer protection is satisfactory

A rapid, simple, and reliable assay to authenticate Peruvian kiwicha (A. caudatus) for food applications

Amaranth has acquired great economic impact as functional food, with species originating from Mexico dominating global trade. In contrast, the Peruvian A. caudatus (kiwicha) has been vastly neglected, although it is endowed with very promising nutritive traits. Morphological plasticity and taxonomic ambiguities render authentication of Amaranth difficult, such that the identity of commercial samples is often unclear. To safeguard the authenticity of kiwicha and, thus, consumer safety, we characterised a germplasm collection of 84 Amaranth accessions on both, the morphological and the genetic level. We show that kiwicha can be delineated phenotypically from other species by its late flowering, taller posture, and lower grain yields. Instead, flower and seed color, often used as proxy for identity, do not qualify as taxonomic markers. Using the plastidic barcoding marker psbA-trnH igs we were able to identify a specific Single Nucleotide Polymorphism (SNP) that separated kiwicha from all other species of Amaranth. This allowed us to develop a sequencing-free authentication assay using an Amplified Refractory Mutation System (ARMS) strategy. As a result kiwicha in commercial samples can be authenticated by a single duplex-PCR yielding a diagnostic side band reporting A. caudatus against all other species of Amaranthus. This fingerprinting assay will help to develop the nutritive potential of kiwicha and to safeguard seed material for A. caudatus against adulteration by the far more prevalent species from Mexico

Cellular Base of Mint Allelopathy: Menthone Affects Plant Microtubules

Plants can use volatiles for remote suppression of competitors. Mints produce essential oils, which are known to affect the growth of other plants. We used a comparative approach to identify allelopathic compounds from different Mints (genus Mentha, but also including Cat Mint, Nepeta cataria, and Corean Mint, Agastache rugosa, belonging to sisters clades within the Mentheae) using the standard cress germination assay as readout. To understand the mechanism behind this allelopathic effect, we investigated the response of tobacco BY-2 cell lines, expressing GFP-tagged markers for microtubules and actin filaments to these essential oils. Based on the comparison between bioactivity and chemical components, we identified menthone as prime candidate for the allelopathic effect, and confirmed this bioactivity targeted to microtubules experimentally in both, plant cells (tobaccoBY-2), and seedlings (Arabidopsis thaliana). We could show that menthone disrupted microtubules and induced mortality linked with a rapid permeabilization (less than 15 min) of the plasma membrane. This mortality was elevated in a tubulin marker line, where microtubules are mildly stabilized. Our study paves the way for the development of novel bioherbicides that would be environmentally friendly

DNA barcoding as new diagnostic tool to lethal plant poisoning in herbivorous mammals

Reliable identification of plant species in the digestive tract of a deceased animal often represents the major key to diagnose a lethal intoxication with poisonous plants in veterinary pathology. In many cases, identification of the species is challenging or even impossible because the diagnostic morphological features have been degraded, and because the interpretation of such features requires a considerable expertise in plant anatomy and biodiversity. The use of DNA barcoding markers can support or even replace classical morphological assessment. While these markers have been widely used for plant taxonomy, their forensic application to clarify causes of animal poisoning is novel. In addition, we use specific single-nucleotide polymorphisms as fingerprints. This allows for a clear decision even in cases, where the conventionally used statistical e-values remain ambiguous. In the current work, we explore the feasibility of this strategy in a couple of exemplary cases, either in concert with anatomical diagnostics, or in cases where visual species identification is not possible, or where chemical toxin detection methods are not well established, complex, time consuming and expensive