The effect of constitutive invertase overexpression on the arbuscular mycorrhiza (AM) is shown. The analysis of the enhanced potential for sucrose cleavage was performed with a heterozygous line of Nicotiana tabacum 35S::cwINV expressing a chimeric gene encoding apoplast‐located yeast‐derived invertase with the CaMV35S promoter. Despite the 35S promoter, roots of the transgenic plants showed no or only minor effects on invertase activity whereas the activity in leaves was increased at different levels. Plants with strongly elevated leaf invertase activity, which exhibited a strong accumulation of hexoses in source leaves, showed pronounced phenotypical effects like stunted growth and chlorosis, and an undersupply of the root with carbon. Moreover, transcripts of PR (pathogenesis related) genes accumulated in the leaves. In these plants, mycorrhization was reduced. Surprisingly, plants with slightly increased leaf invertase activity showed a stimulation of mycorrhization, particularly 3 weeks after inoculation. Compared with wild‐type, a higher degree of mycorrhization accompanied by a higher density of all fungal structures and a higher level of Glomus intraradices ‐specific rRNA was detected. Those transgenic plants showed no accumulation of hexoses in the source leaves, minor phenotypical effects and no increased PR gene transcript accumulation. The roots had even lower levels of phenolic compounds (chlorogenic acid and scopolin), amines (such as tyramine, dopamine, octopamine and nicotine) and some amino acids (including 5‐amino‐valeric acid and 4‐amino‐butyric acid), as well as an increased abscisic acid content compared with wild‐type. Minor metabolic changes were found in the leaves of these plants. The changes in metabolism and defense status of the plant and their putative role in the formation of an AM symbiosis are discussed.

Two new groups of cholane-peptoid hybrid macrocycles were produced by implementing novel combinations of the MiB methodology. Steroid-based hybrid macrolactams including heterocycle and aryl moieties were obtained by utilizing cholanic dicarboxylic acids and diamines in a bidirectional double Ugi-Four-Component (Ugi-4CR) based macrocyclization protocol. Alternatively, N-substituted cyclocholamides were produced from a cholanic pseudo-amino acid by an Ugi-4CR-based cyclooligomerization approach. Both types of macrocycles are steroid-peptoid hybrid macrocycles containing exocyclic peptidic chains. These novel frameworks are a result of the use of bile acids bifunctionalized with carboxylic and amino functionalities as bifunctional building blocks of the Ugi-MiB approach.

A detailed study of the Baeyer-Villiger reaction of 3-ketosteroids has been performed by using m-chloroperoxybenzoic and trifluoroperoxyacetic acids as oxidants. The process was fully regiospecific for 3-keto-5α-steroids with the employ of both peracids, and only partially regioselective for 3-keto-5β-steroids by using trifluoroperoxyacetic acid. Interestingly, the reaction resulted completely unselective for 3-keto-5β-steroids by using m-chloroperoxybenzoic acid. Theoretical studies were performed to explain the regiochemistry of this process, which is suggested to be controlled by conformational effects in the transition state of the Criegee rearrangement.

Small libraries of peptoid-based macro(multi)cycles were produced by applying combinatorial principles to the MiB methodology. This combinatorial approach features the incorporation of varied building blocks into the resulting macrocycles by mixing all the components in the same reaction pot. Both skeletal and appendage diversity could be generated in one shot due to the multicomponent nature of the system. HPLC and ESI-MS analyses showed a well-distributed composition of the libraries and revealed the presence of all possible macrocycles resulting from the different combinations of building blocks, especially of members with differentially substituted bridges not available by any other one-pot ­approach.

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Conifers produce terpenoid-based oleoresins as constitutive and inducible defenses against herbivores and pathogens. Much information is available about the genes and enzymes of the late steps of oleoresin terpenoid biosynthesis in conifers, but almost nothing is known about the early steps which proceed via the methylerythritol phosphate (MEP) pathway. Here we report the cDNA cloning and functional identification of three Norway spruce (Picea abies) genes encoding 1-deoxy-d-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, and their differential expression in the stems of young saplings. Among them are representatives of both types of plant DXS genes. A single type I DXS gene is constitutively expressed in bark tissue and not affected by wounding or fungal application. In contrast, two distinct type II DXS genes, PaDXS2A and PaDXS2B, showed increased transcript abundance after these treatments as did two other genes of the MEP pathway tested, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 4-hydroxyl 3-methylbutenyl diphosphate reductase (HDR). We also measured gene expression in a Norway spruce cell suspension culture system that, like intact trees, accumulates monoterpenes after treatment with methyl jasmonate. These cell cultures were characterized by an up-regulation of monoterpene synthase gene transcripts and enzyme activity after elicitor treatment, as well as induced formation of octadecanoids, including jasmonic acid and 12-oxophytodienoic acid. Among the Type II DXS genes in cell cultures, PaDXS2A was induced by treatment with chitosan, methyl salicylate, and Ceratocystis polonica (a bark beetle-associated, blue-staining fungal pathogen of Norway spruce). However, PaDXS2B was induced by treatment with methyl jasmonate and chitosan, but was not affected by methyl salicylate or C. polonica. Our results suggest distinct functions of the three DXS genes in primary and defensive terpenoid metabolism in Norway spruce.

There is considerable evidence suggesting that jasmonates (JAs) play a role in plant resistance against abiotic stress. It is well known that in Angiosperms JAs are involved in the defense response, however there is little information about their role in Gymnosperms. Our proposal was to study the involvement of JAs in Pinus pinaster Ait. reaction to cold and water stress, and to compare the response of two populations of different provenances (Gredos and Bajo Tiétar) to these stresses. We detected 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and the hydroxylates 11-hydroxyjasmonate and 12-hydroxyjasmonate in foliage and shoots of P. pinaster plants. The response of the Gredos population to cold stress differed from that of Bajo Tiétar. Gredos plants showed a lower JA-basal level than Bajo Tiétar; under cold stress JA increased twofold at 72 h, while it decreased in Bajo Tiétar plants. The hydroxylates slightly increased in both populations due to cold stress treatment. Under water stress, plants from Gredos showed a remarkable JA-increase; thus the JA-response was much more prominent under water stress than under cold stress. In contrast, no change was found in JA-level in Bajo Tiétar plants under water stress. The level of JA-precursor, OPDA, was very low in control plants from Gredos and Bajo Tiétar. Under water stress OPDA increased only in plants from Bajo Tiétar. Therefore, we inform here of a different JAs-accumulation pattern after the stress treatment in P. pinaster from two provenances, and suggest a possible correlation with adaptations to diverse ecological conditions.

The catalytic enantioselective arylation of several aldehydes using arylboronic acids as the source of transferable aryl groups is described; the reaction is found to proceed in excellent yields and high enantioselectivities (up to 96% ee) in the presence of a chiral amino alcohol derived from ephedrines and congeners.

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Geranylgeraniol (GGOH) is an acyclic diterpene that posesses apoptotic activity to cancer cells [1]. It has been proposed to be the main intermediate of the biosynthetic pathway of plaunotol, an antipeptic ulcer drug from Croton stellatopilosus [2]. Our enzymological studies showed that GGOH is formed from the dephosphorylation of geranylgeranyl pyrophosphate (GGPP), through sequential monodephosphorylation [3], by the action of GGPP phosphatase enzyme [4]. As part of our interest in manipulating the gene of GGPP phosphatase for the production of GGOH in Escherichia coli system, we began with cloning of cDNA encoding prenyl diphosphate phosphatase from C. stellatopilosus. The degenerated primers were designed from the alignment of amino acid sequences of prenyl diphosphate phosphatase in database. The full-length gene was obtained by RACE-PCR. The cDNA contained an open reading frame encoding 888 amino acids with a calculated molecular mass of 33.6 kDa. The phosphatase motif [5] was included in the deduced amino acid sequence consisting of KX6RP, PSGH, and SRX5HX3D. Its amino acid sequence showed 71% identity to phosphatidic acid phosphatase from Vigna unguiculata. The topology prediction of the enzyme indicated that it was a transmembrane protein with 6 transmembrane regions. The recombinant prenyl diphosphate phosphatase and its 4 designed truncated genes were expressed in Escherichia coli BL21(DE3)RIL. Detection of their phosphatase activities by using [1-3H]GGPP and farnesyl pyrophosphate ([1-3H]FPP) as substrates showed that their enzymatic products of [1-3H]GGOH and [1-3H]FOH, respectively, were formed in the assay mixture. The results suggested the potential of GGOH production by the recombinant E. coli although the expression of the recombinant gene was still in low level.