Jingjing Fang

Tissue-Specific Distribution of Secondary Metabolites in Rapeseed (Brassica napus L.)

Four different parts, hypocotyl and radicle (HR), inner cotyledon (IC), outer cotyledon (OC), seed coat and endosperm (SE), were sampled from mature rapeseed (Brassica napus L.) by laser microdissection. Subsequently, major secondary metabolites, glucosinolates and sinapine, as well as three minor ones, a cyclic spermidine conjugate and two flavonoids, representing different compound categories, were qualified and quantified in dissected samples by high-performance liquid chromatography with diode array detection and mass spectrometry. No qualitative and quantitative difference of glucosinolates and sinapine was detected in embryo tissues (HR, IC and OC). On the other hand, the three minor compounds were observed to be distributed unevenly in different rapeseed tissues. The hypothetic biological functions of the distribution patterns of different secondary metabolites in rapeseed are discussed.

Laser microdissection: a sample preparation technique for plant micrometabolic profiling

INTRODUCTION Unlike unicellular organisms, plants have evolved as complex organisms that are defined by their ability to distribute special vital functions to spatially separated organs and tissues. Current phytochemical approaches mostly ignore this fact by analysing samples that consist of different cell types and thus average the information obtained. A comprehensive metabolite analysis with high spatial resolution is essential to fully characterise the state of a certain tissue; hence, the analysis of metabolites occurring in specialised plant cells is of considerable interest in chemical ecology, plant natural product chemistry and other bioscience disciplines. Laser microdissection (LMD), including laser capture microdissection and laser microdissection and pressure catapulting, is a convenient sampling technique to harvest homogeneous cell types for the microanalysis of plant metabolites. OBJECTIVE The objective of this work is to provide an introduction to LMD methodology and a concise review of recent applications of LMD in the high-resolution analysis of metabolites in different plant materials. METHODS A step-by-step approach to LMD sampling techniques is described. How LMD can be used to sample cells or microscopic tissue pieces from different plant organs, such as leaves, stems, and seeds, is shown in detail. Finally, the future of LMD in plant metabolites analysis is discussed. RESULTS This review summarises studies over the past decade not only showing technical details but also indicating the wide application of this method for high-resolution plant metabolite analysis. CONCLUSION Laser microdissection is a powerful sampling technique for plant micrometabolic profiling and metabolomics research.

Phytochemical profile of aerial parts and roots of Wachendorfia thyrsiflora L. studied by LC-DAD-SPE-NMR

Hyphenated liquid chromatography - diode array detection - solid phase extraction - nuclear magnetic resonance spectroscopy (LC-DAD-SPE-NMR) was used to investigate the phytochemical composition of aerial parts and roots of Wachendorfia thyrsiflora (Haemodoraceae). Eleven phenylphenalenones and related compounds were identified in the aerial parts of the plant, ten compounds were found in the roots, and four additional compounds occurred in both plant parts. Twelve compounds are previously unreported natural products including five alkaloids (phenylbenzoisoquinolinones) are described here for the first time. In the work presented here, phenylphenalenones with an intact C(19) core structure were found only in the roots. Oxa analogs with a C(18)O scaffold occurred both in the roots and in the aerial plant parts, while most of the aza analogs with a C(18)N scaffold were detected in the aerial plant parts. This distribution pattern suggests that phenylphenalenones form in the roots, then the intact C(19) skeleton is converted into oxa analogs in the roots, translocated into the leaves and further reacted with amines or amino acids to form aza analogs (phenylbenzoisoquinolin-1,6-dione alkaloids).

Development of an NMR metabolomics-based tool for selection of flaxseed varieties

Flaxseed is an important source of lignans and ω-3 fatty acids, compounds which present interest in human health with many applications in food industry. It is therefore necessary to precisely know the metabolite content in flaxseed. A metabolomic approach using NMR was developed to achieve this goal. Due to particular characteristics of flaxseed (high level in oil, high amount in mucilage, and integration of the phenolics into a macromolecule), the extraction procedure had first to be optimized using an experimental design, based on the extraction time, in a water bath or an ultrasound bath, alkaline treatment, defatting, and centrifugation temperature. This methodology was then applied to several flaxseed varieties classified in function of their content in ω-3 fatty acid. The main differences in semi-polar metabolites between these varieties concern compounds of the phenylpropanoid pathway. Hydroxycinnamic acid glucoside and lignan content increase when ω-3 fatty acid content decrease whereas flavonoid content increase in the same way of ω-3 fatty acids.

Co-occurrence of phenylphenalenones and flavonoids in Xiphidium caeruleum Aubl. flowers

A Xiphidium caeruleum flower extract was separated by semi-preparative HPLC into five fractions, from which three flavonoids, two phenylphenalenones and 17 phenylphenalenone-related compounds including five unknown compounds, were isolated and their structures elucidated by Liquid Chromatography-Diode Array Detection-Solid Phase Extraction-Nuclear Magnetic Resonance spectroscopy (LC-DAD-SPE-NMR) and mass spectrometry (MS). This is the first report of the co-occurrence of phenylphenalenones and flavonoids in the Haemodoraceae family. The ecological implications of flavonoids and various phenylphenalenone-type compounds and their putative biosynthesis sites in X. caeruleum are subject to discussion.

Concentration Kinetics of Secoisolariciresinol Diglucoside and its Biosynthetic Precursor Coniferin in Developing Flaxseed

INTRODUCTION In the plant kingdom, flaxseed (Linum usitatissimum L.) is the richest source of secoisolariciresinol diglucoside (SDG), which is of great interest because of its potential health benefits for human beings. The information about the kinetics of SDG formation during flaxseed development is rare and incomplete. OBJECTIVE In this study, a reversed-phase high-performance liquid chromatography-diode array detection (HPLC-DAD) method was developed to quantify SDG and coniferin, a key biosynthetic precursor of SDG in flaxseed. METHODOLOGY Seeds from different developmental stages, which were scaled by days after flowering (DAF), were harvested. After alkaline hydrolysis, the validated HPLC method was applied to determine SDG and coniferin concentrations of flaxseed from different developing stages. RESULTS Coniferin was found in the entire capsule as soon as flowering started and became undetectable 20 DAF. SDG was detected 6 DAF, and the concentration increased until maturity. On the other hand, the SDG amount in a single flaxseed approached the maximum around 25 DAF, before desiccation started. Concentration increase between 25 DAF and 35 DAF can be attributed to corresponding seed weight decrease. CONCLUSION The biosynthesis of coniferin is not synchronous with that of SDG. Hence, the concentrations of SDG and coniferin change during flaxseed development.

Laser microdissection and spatiotemporal pinoresinol-lariciresinol reductase gene expression assign the cell layer-specific accumulation of secoisolaricirésinol diglucoside in flaxseed coats

The concentration of secoisolariciresinol diglucoside (SDG) found in flaxseed (Linum usitatissimum L.) is higher than that found in any other plant. It exists in flaxseed coats as an SDG-3-hydroxy-3-methylglutaric acid oligomer complex. A laser microdissection method was applied to harvest material from different cell layers of seed coats of mature and developing flaxseed to detect the cell-layer specific localization of SDG in flaxseed; NMR and HPLC were used to identify and quantify SDG in dissected cell layers after alkaline hydrolysis. The obtained results were further confirmed by a standard molecular method. The promoter of one pinoresinol-lariciresinol reductase gene of L. usitatissimum (LuPLR1), which is a key gene involved in SDG biosynthesis, was fused to a β-glucuronidase (GUS) reporter gene, and the spatio-temporal regulation of LuPLR1 gene expression in flaxseed was determined by histochemical and activity assays of GUS. The result showed that SDG was synthesized and accumulated in the parenchymatous cell layer of the outer integument of flaxseed coats.