Engineering a highly sensitive biosensor for abscisic acid in mammalian cells.

Abscisic acid (ABA) is a signalling molecule conserved in plants, bacteria, fungi, and animals. Recently, ABA has gained attention for its pharmacological activities and its potential as a biomarker for the severity of chronic obstructive pulmonary disease and glioma. This prompts the development of a reliable, sensitive, rapid, and cost-effective method to quantify ABA levels in mammalian cells and tissues. The previously described ABA biosensor system based on the ABA-dependent interaction between the plant ABA receptor PYL1 and co-receptor ABI1 is not sensitive enough for the low ABA levels seen in mammals. Therefore, we optimized this system by replacing PYL1 with other high-affinity plant PYL proteins. The optimized biosensor system engineered with the PYL8 receptor enabled the quantification of ABA at low concentrations in HEK293T cells.

Modulation of Arabidopsis root growth by specialized triterpenes.

Plant roots are specialized belowground organs that spatiotemporally shape their development in function of varying soil conditions. This root plasticity relies on intricate molecular networks driven by phytohormones, such as auxin and jasmonate (JA). Loss-of-function of the NOVEL INTERACTOR OF JAZ (NINJA), a core component of the JA signaling pathway, leads to enhanced triterpene biosynthesis, in particular of the thalianol gene cluster, in Arabidopsis thaliana roots. We have investigated the biological role of thalianol and its derivatives by focusing on Thalianol Synthase (THAS) and Thalianol Acyltransferase 2 (THAA2), two thalianol cluster genes that are upregulated in the roots of ninja mutant plants. THAS and THAA2 activity was investigated in yeast, and metabolite and phenotype profiling of thas and thaa2 loss-of-function plants was carried out. THAA2 was shown to be responsible for the acetylation of thalianol and its derivatives, both in yeast and in planta. In addition, THAS and THAA2 activity was shown to modulate root development. Our results indicate that the thalianol pathway is not only controlled by phytohormonal cues, but also may modulate phytohormonal action itself, thereby affecting root development and interaction with the environment.

Avocado kernels, an industrial residue: a source of compounds with insecticidal activity against silverleaf whitefly.

Fruit processing waste, such as kernels (endocarp + seed) of avocado [Persea americana Mill. (Lauraceae)], could be used as raw material in the preparation of botanical insecticides. In light of this potential, this study assessed the insecticidal action of extracts and fractions from kernels of two avocado cultivars (Breda and Margarida) on Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype B, an important pest species in tropical conditions. Ethanolic and aqueous extracts prepared from kernels of P. americana, regardless of the plant cultivar used, caused promising insecticidal activity to whitefly nymphs. Based on yield in crude extracts [10.32 and 9.85% (w/w), respectively, for cultivars Breda and Margarida], on the bioassay results with crude extracts and on the chemical profiles, the ethanolic extract of kernels of P. americana cv. Breda was chose for the continuation of the study. Thus, the ethanolic extract of kernels of cv. Breda (LC50 = 197.84 ppm and LC90 = 567.19 ppm) was selected and subjected to fractionation by the liquid-liquid partition technique. The hexane and dichloromethane fractions of this extract caused significant mortality of nymphs. The analysis using the ultraviolet (UV) and hydrogen nuclear magnetic resonance (1H NMR) showed the presence of long-chain aliphatic compounds (alkanols or acetogenins of Lauraceae), alkylfurans (or avocadofurans), and unsaturated fatty acids in these fractions, which are possibly related to bioactivity observed in B. tabaci, besides saccharides. The results show that kernels of P. americana are promising sources of compounds with insecticidal action for the control of B. tabaci biotype B, a great opportunity to transform environmental problems into eco-friendly solutions to agriculture.

A Seed-Specific Regulator of Triterpene Saponin Biosynthesis in Medicago truncatula.

Plants produce a vast array of defense compounds to protect themselves from pathogen attack or herbivore predation. Saponins are a specific class of defense compounds comprising bioactive glycosides with a steroidal or triterpenoid aglycone backbone. The model legume Medicago truncatula synthesizes two types of saponins, hemolytic saponins and nonhemolytic soyasaponins, which accumulate as specific blends in different plant organs. Here, we report the identification of the seed-specific transcription factor TRITERPENE SAPONIN ACTIVATION REGULATOR3 (TSAR3), which controls hemolytic saponin biosynthesis in developing M. truncatula seeds. Analysis of genes that are coexpressed with TSAR3 in transcriptome data sets from developing M. truncatula seeds led to the identification of CYP88A13, a cytochrome P450 that catalyzes the C-16α hydroxylation of medicagenic acid toward zanhic acid, the final oxidation step of the hemolytic saponin biosynthesis branch in M. truncatula In addition, two uridine diphosphate glycosyltransferases, UGT73F18 and UGT73F19, which glucosylate hemolytic sapogenins at the C-3 position, were identified. The genes encoding the identified biosynthetic enzymes are present in clusters of duplicated genes in the M. truncatula genome. This appears to be a common theme among saponin biosynthesis genes, especially glycosyltransferases, and may be the driving force of the metabolic evolution of saponins.

CYP712K4 Catalyzes the C-29 Oxidation of Friedelin in the Maytenus ilicifolia Quinone Methide Triterpenoid Biosynthesis Pathway.

The native Brazilian plant Maytenus ilicifolia accumulates a set of quinone methide triterpenoids with important pharmacological properties, of which maytenin, pristimerin and celastrol accumulate exclusively in the root bark of this medicinal plant. The first committed step in the quinone methide triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene to friedelin, catalyzed by the oxidosqualene cyclase friedelin synthase (FRS). In this study, we produced heterologous friedelin by the expression of M. ilicifolia FRS in Nicotiana benthamiana leaves and in a Saccharomyces cerevisiae strain engineered using CRISPR/Cas9. Furthermore, friedelin-producing N. benthamiana leaves and S. cerevisiae cells were used for the characterization of CYP712K4, a cytochrome P450 from M. ilicifolia that catalyzes the oxidation of friedelin at the C-29 position, leading to maytenoic acid, an intermediate of the quinone methide triterpenoid biosynthesis pathway. Maytenoic acid produced in N. benthamiana leaves was purified and its structure was confirmed using high-resolution mass spectrometry and nuclear magnetic resonance analysis. The three-step oxidation of friedelin to maytenoic acid by CYP712K4 can be considered as the second step of the quinone methide triterpenoid biosynthesis pathway, and may form the basis for further discovery of the pathway and heterologous production of friedelanes and ultimately quinone methide triterpenoids.

Scope of the 2(5H)-furanone helicity rule: a combined ECD, VCD, and DFT investigation.

One of the most widely used methods to assess the stereochemistry of chiral 2(5H)-furanones is an empirical electronic circular dichroism (ECD) helicity rule. In the present work, an extensive experimental and theoretical investigation of the scope of the above-mentioned empirical rule for acetogenins with a hydroxyl group substituted at C-4 revealed a possible exception to this rule. The underlying causes for this observation are discussed with respect to side chain substitutions, conformational requirements, chromophore handedness as well as a qualitative orbital analysis. Further investigation using vibrational circular dichroism (VCD) spectroscopy led to the identification of spectral markers that seem to be more localized and less affected by side chain substitutions. As the presence of a [capital Upsilon]-lactone ring and a hydroxyl group at C-4 is a very common structural feature of Annonaceous acetogenins, we recommend the combined use of ECD and VCD spectroscopy, along with quantum chemical computations, for the stereochemical analysis of structurally related molecules.