Another Route for Amino Acid Production?: Reverse Genetic Probing for a Functional Cytosolic Shikimate Pathway in Plants

The shikimate pathway is a metabolic pathway that produces the three aromatic amino acids—phenylalanine, tryptophan, and tyrosine—which are essential to human diets and necessary for many plant functions. Consequently, the shikimate pathway is commonly targeted for antibiotic and herbicide strategies as well as genetic engineering in several fields. This pathway is known to be localized in the plastids, or double membrane-bound organelles, of plant cells; however, there is enzymatic evidence of another shikimate pathway in the cell fluid, or cytosol. To determine whether a complete cytosolic shikimate pathway exists, we used a modified gene for the first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, that will be produced exclusively in the cytosol. We inserted this gene into both Petunia hybrida cv. multiflora and Petunia hybrida cv. Mitchell, which produce high levels of phenylalanine-derived volatile organic compounds (VOCs). We will determine the gene’s effect by measuring the change in the rate of production of the VOCs, which corresponds to overall activity of the shikimate pathway. Therefore, if there is a fully-functional, cytosolic shikimate pathway, this rate will increase compared to wild-type petunias. We are also investigating putative cytosolic, shikimate pathway enzymes 3-dehydroquinate synthase and 3-dehydroquinate dehydratase/shikimate dehydrogenase. We will determine their subcellular localization by performing confocal microscopy on green fluorescent protein variants and their function by interfering RNA suppression. While further investigation is required, this cytosolic function could provide a new, innovative target for antibiotics, herbicides, and genetic engineering

The Effect of Transient HMG-CoA Reductase and 1-Deoxy-D-Xylulose-5-Phosphate Synthase Overexpression on Terpene Production in Transgenic Tomato Fruits

Isoprenoids are secondary metabolites that control numerous plant functions including signaling, growth, photosynthesis, and membrane structure. The bioengineering of isoprenoid synthesis could produce plants with a variety of beneficial traits. Plants form isoprenoids using two different pathways, the mevalonate (MVA) pathway and the methylerithritol phosphate (MEP) pathway, which cooperate via metabolic cross-talk. Transgenic tomato lines expressing both the plastidic and cytosolic forms of the snapdragon nerolidol/linalool terpene synthase under a fruit ripening specific promoter were transiently transformed to overexpress key enzymes in the two isoprenoid pathways. Hydroxymethylglutaryl-coenzyme A reductase (HMGR) is the rate limiting enzyme in the MVA pathway that was selected for overexpression. 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) in the plastid was targeted as it is the first committed step in the MEP pathway. HMGR and DXPS coding regions were cloned into binary vectors under a constitutive promoter and introduced into Agrobacterium tumefaciens which were then injected into ripening tomato fruits for transient expression. Additionally, untransformed fruits were incubated with either the MVA-inhibitor mevinolin or the MEP-inhibitor fosmidomycin. Terpene production was characterized by gas chromatography and mass spectrometery of fruit volatiles collected at the ripe stage. Inhibitor treatment is expected to decrease terpene synthesis in the same compartment as the inhibited pathway. The overexpression of early MVA and MEP pathway genes is expected to significantly increase the formation of terpenes

The effect of an Enhanced Isopentenyl Monophosphate Pool on Terpenoid Biosynthesis in vivo

Found in all living organisms, terpenoids make up the largest group of natural products and are essential compounds for many major processes, including photosynthesis, respiration, hormone production, and electron transport. Additionally, they have commercial and medical value in products including fragrances, cosmetics, and medicines. Terpenoids originate from the five-carbon building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are synthesized by the mevalonic acid (MVA) and methylerithritol phosphate (MEP) pathways. An alternative MVA pathway was discovered in Archaea with the final two enzymes being phosphomevalonate decarboxylase (MPD) and isopentenyl phosphate kinase (IPK). Even though this alternative pathway is not present in plants, presence of IPK was retained. The overexpression of IPK in planta indicates that IPK plays a significant role in the MVA pathway by synthesizing IPP/DMAPP from an IP/DMAP pool for terpenoid biosynthesis. It has been suggested that this monophosphate pool regulates downstream carbon flux by inhibiting farnesyl diphosphate synthase (FPPS). By utilizing MPD from the archeabacterium Roseiflexus castenholzii, we can see how an increased isopentenyl (IP) pool affects downstream terpenoid biosynthesis. To do this, RcMPD was overexpressed in the background of Arabidopsis thaliana T-DNA insertion lines of a knockdown of IPK. These lines were tested for expression of MPD/IPK using qrt-PCR and terpenoids were analyzed via sterol extraction and scent collection. Levels of monoterpenes (MEP pathway products) and sesquiterpenes (MVA pathway products) were significantly reduced, suggesting that a larger monophosphate pool reduces downstream synthesis of farnesyl diphosphate, a precursor for sterol and sesquiterpene biosynthesis

The link between language and culture on the lessons of Russian as a foreign language

The main aim of a foreign language teacher is to form a student’s communicative competence, which is a complex of other competencies such as linguistic, discursive and linguocultural.  For successful psychological and social adaptation in a new cultural and linguistic space for a foreign student is extremely important at the initial level of education begin to master the basic linguistic and cultural concepts that reflect the culture of the speakers of the studied language and leads to the adoption of a different worldview.  Thus, for successful communication, you need not only use phonetic, grammatical, syntactic and pragmatic rules of the language, but also you should have a clear idea of the conceptual picture of the world of the people, who speaks this language.  It follows that the study of any foreign language should occur inextricably linked with the knowledge of culture, values and understanding of the native people of this language. The objective of the work is to formulate the key linguocultural principles of teaching Russian as a foreign language. To achieve this objective, the works of leading researchers in the field of linguistics, didactics, methods of teaching Russian as a foreign language have been analyzed. The research object is an inextricable link between learning a foreign language and the culture of its speakers. The research result is the proof of the need to learn a foreign language as being inextricably linked with knowledge of the culture, values and world outlook of the people - speakers of this language, as well as a list of basic linguocultural principles, on which teaching a foreign language, including Russian as a foreign language, should be based

Культурно-цивилизационный феномен интеллигенции: историческая и провидческая реальность

The article attempts to consider the phenomenon of the Russian intelligentsia in a wide cultural context. We turn to the works of Russian philosophers, to the works of art by Russian writers. Philosophy, literature and art in Russian culture form a synthesis through which you can understand the uniqueness of the national image of the world. The material for the study is the treatise S.A. Yesenin "The keys of Mary”. In this treatise, the poet reflects not only on the laws of art, but also gives ideas about the Russian man, his connection with archaic knowledge, expressed through ornament, embroidery, and applied art. The comparative-typological, structural-functional and systematic analysis method is applied.El artículo intenta considerar el fenómeno de la intelectualidad rusa en un amplio contexto cultural. Pasamos a las obras de filósofos rusos, a las obras de arte de escritores rusos. La filosofía, la literatura y el arte en la cultura rusa forman una síntesis a través de la cual puedes entender la singularidad de la imagen nacional del mundo. El material para el estudio es el tratado SA Yesenin "Las llaves de María". En este tratado, el poeta no solo reflexiona sobre las leyes del arte, sino que también da ideas sobre el hombre ruso, su conexión con el conocimiento arcaico, expresada a través del ornamento. , bordado y arte aplicado Se aplica el método de análisis comparativo-tipológico, estructural-funcional y sistemático.В статье предпринимается попытка рассмотреть феномен русской интеллигенции в широком культурном контексте. Обращаемся к трудам русских философов, к художественным произведениям русских писателей. Философия, литература и искусство в русской культуре образуют синтез, через который можно понять своеобразие национального образа мира. Материалом для исследования выступает трактат С.А. Есенина «Ключи Марии». В этом трактате поэт размышляет не только о законах искусства, но и дает представления о русском человеке, его связи с архаическими знаниями, выразившейся через орнамент, вышивку, прикладное искусство. Применен сравнительно-типологический, структурно-функциональный и системный метод анализа

A Survey of Oxidative Paracatalytic Reactions Catalyzed by Enzymes That Generate Carbanionic Intermediates: Implications for ROS Production, Cancer Etiology, and Neurodegenerative Diseases

Enzymes that generate carbanionic intermediates often catalyze paracatalytic reactions with O2 and other electrophiles not considered “normal” reactants. For example, pyridoxal 5′-phosphate (PLP)—containing pig kidney dopa decarboxylase oxidizes dopamine with molecular O2 to 3,4-dihydroxyphenylacetaldehyde at about 1% of the rate at which it catalyzes nonoxidative dopa decarboxylation. The mutant Y332F enzyme, however, catalyzes stoichiometric conversion of dopa to 3,4-dihydroxyphenylacetaldehyde, suggesting that even minor structural changes may alter or initiate paracatalytic reactions catalyzed by certain enzymes. Carbanions generated by several thiamine diphosphate (ThDP)—dependent enzymes react with different electrophiles, transforming some xenobiotics and endogenous compounds into potentially biologically hazardous products. The detrimental effects of paracatalytic reactions may be greatly increased by cellular compartmentation of enzymes and intermediates. For example, in two of the the three multienzyme complexes involved in oxidative α-keto acid decarboxylation, paracatalytic reactions of the third component inactivate the first carbanion-generating component. In this review we provide an outline of carbanion-generating enzymes known to catalyze paracatalytic reactions. We also discuss the potential of some of these reactions to contribute to irreversible damage in cancer and neurodegeneration through disease-induced alterations in the metabolic state and/or protein structure

Adaptive mechanisms of plant specialized metabolism connecting chemistry to function

As sessile organisms, plants evolved elaborate metabolic systems that produce a plethora of specialized metabolites as a means to survive challenging terrestrial environments. Decades of research have revealed the genetic and biochemical basis for a multitude of plant specialized metabolic pathways. Nevertheless, knowledge is still limited concerning the selective advantages provided by individual and collective specialized metabolites to the reproductive success of diverse host plants. Here we review the biological functions conferred by various classes of plant specialized metabolites in the context of the interaction of plants with their surrounding environment. To achieve optimal multifunctionality of diverse specialized metabolic processes, plants use various adaptive mechanisms at subcellular, cellular, tissue, organ and interspecies levels. Understanding these mechanisms and the evolutionary trajectories underlying their occurrence in nature will ultimately enable efficient bioengineering of desirable metabolic traits in chassis organisms

Petunia × hybrida floral scent production is negatively affected by high‐temperature growth conditions

Increasing temperatures due to changing global climate are interfering with plant–pollinator mutualism, an interaction facilitated mainly by floral colour and scent. Gas chromatography–mass spectroscopy analyses revealed that increasing ambient temperature leads to a decrease in phenylpropanoid‐based floral scent production in two Petunia × hybrida varieties, P720 and Blue Spark, acclimated at 22/16 or 28/22 °C (day/night). This decrease could be attributed to down‐regulation of scent‐related structural gene expression from both phenylpropanoid and shikimate pathways, and up‐regulation of a negative regulator of scent production, emission of benzenoids V (EOBV). To test whether the negative effect of increased temperature on scent production can be reduced in flowers with enhanced metabolic flow in the phenylpropanoid pathway, we analysed floral volatile production by transgenic ‘Blue Spark’ plants overexpressing CaMV 35S‐driven Arabidopsis thaliana production of anthocyanin pigments 1 (PAP1) under elevated versus standard temperature conditions. Flowers of 35S:PAP1 transgenic plants produced the same or even higher levels of volatiles when exposed to a long‐term high‐temperature regime. This phenotype was also evident when analysing relevant gene expression as inferred from sequencing the transcriptome of 35S:PAP1 transgenic flowers under the two temperature regimes. Thus, up‐regulation of transcription might negate the adverse effects of temperature on scent production.We demonstrate that petunia flowers produce less volatile phenylpropanoid compounds, in both scent bouquets and internal pools, in response to elevated temperatures. We reveal that the decrease in floral scent is correlated with reduced transcript levels of scent‐related genes, and that the adverse effect of high temperature can be negated by expressing transcriptional up‐regulators. We believe that the conclusions and implications drawn from the original data presented in our manuscript will be of particular interest to a broad spectrum of your readers, particularly in view of recent changes in global climate and the risk of environmental disruption of plant–pollinator mutualism.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/112003/1/pce12486-sup-0001-si.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/112003/2/pce12486.pd

Interlinking showy traits: co-engineering of scent and colour biosynthesis in flowers

The phenylpropanoid pathway gives rise to metabolites that determine floral colour and fragrance. These metabolites are one of the main means used by plants to attract pollinators, thereby ensuring plant survival. A lack of knowledge about factors regulating scent production has prevented the successful enhancement of volatile phenylpropanoid production in flowers. In this study, the Production of Anthocyanin Pigment1 ( Pap1 ) Myb transcription factor from Arabidopsis thaliana , known to regulate the production of non-volatile phenylpropanoids, including anthocyanins, was stably introduced into Petunia hybrida . In addition to an increase in pigmentation, Pap1 -transgenic petunia flowers demonstrated an increase of up to tenfold in the production of volatile phenylpropanoid/benzenoid compounds. The dramatic increase in volatile production corresponded to the native nocturnal rhythms of volatile production in petunia. The application of phenylalanine to Pap1 -transgenic flowers led to an increase in the otherwise negligible levels of volatiles emitted during the day to nocturnal levels. On the basis of gene expression profiling and the levels of pathway intermediates, it is proposed that both increased metabolic flux and transcriptional activation of scent and colour genes underlie the enhancement of petunia flower colour and scent production by Pap1 . The co-ordinated regulation of metabolic steps within or between pathways involved in vital plant functions, as shown here for two showy traits determining plant–pollinator interactions, provides a clear advantage for plant survival. The use of a regulatory factor that activates scent production creates a new biotechnological strategy for the metabolic architecture of fragrance, leading to the creation of novel genetic variability for breeding purposes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75040/1/j.1467-7652.2008.00329.x.pd

Developmental Changes in the Metabolic Network of Snapdragon Flowers

Evolutionary and reproductive success of angiosperms, the most diverse group of land plants, relies on visual and olfactory cues for pollinator attraction. Previous work has focused on elucidating the developmental regulation of pathways leading to the formation of pollinator-attracting secondary metabolites such as scent compounds and flower pigments. However, to date little is known about how flowers control their entire metabolic network to achieve the highly regulated production of metabolites attracting pollinators. Integrative analysis of transcripts and metabolites in snapdragon sepals and petals over flower development performed in this study revealed a profound developmental remodeling of gene expression and metabolite profiles in petals, but not in sepals. Genes up-regulated during petal development were enriched in functions related to secondary metabolism, fatty acid catabolism, and amino acid transport, whereas down-regulated genes were enriched in processes involved in cell growth, cell wall formation, and fatty acid biosynthesis. The levels of transcripts and metabolites in pathways leading to scent formation were coordinately up-regulated during petal development, implying transcriptional induction of metabolic pathways preceding scent formation. Developmental gene expression patterns in the pathways involved in scent production were different from those of glycolysis and the pentose phosphate pathway, highlighting distinct developmental regulation of secondary metabolism and primary metabolic pathways feeding into it