Plant Science

Plant vacuoles are the largest compartment in plant cells, occupying more than 80% of the cell volume. A variety of proteins, sugars, pigments and other metabolites are stored in these organelles (Paris et al., 1996; Olbrich et al., 2007). Flowers produce a variety of specialized metabolites, some of which are unique to this organ, such as components of pollination syndromes, i.e., scent volatiles and flavonoids (Hoballah et al., 2007; Cna'ani et al., 2015). To study the compounds stored in floral vacuoles, this compartment must be separated from the rest of the cell. To enable isolation of vacuoles, protoplasts were first generated by incubating pierced corollas with cellulase and macrozyme enzymes. After filtering and several centrifugation steps, protoplasts were separated from the debris and damaged/burst protoplasts, as revealed by microscopic observation. Concentrated protoplasts were lysed, and vacuoles were extracted by Ficoll-gradient centrifugation. Vacuoles were used for quantitative GC-MS analyses of sequestered metabolites. This method allowed us to identify vacuoles as the subcellular accumulation site of glycosylated volatile phenylpropanoids and to hypothesize that conjugated scent compounds are sequestered in the vacuoles en route to the headspace (Cna'ani et al., 2017).

Metabolite profiling using gas chromatography-mass spectrometry (GC-MS) permits the annotation and quantification of a relatively wide variety of metabolites, covering a wide range of biochemical groups of metabolites. Lisec et al. (2006) established a method for GC-MS profiling in plants. Based on this protocol, we provide here a detailed GC-MS-based metabolite profiling protocol to identify compounds belonging to several biochemical groups in the primary metabolism of mature Arabidopsis thaliana seeds (Cohen et al., 2014). The protocol uses methoxyamine hydrochloride and N-methyl-N-trimethylsilyltriflouroacetamide (MSTFA) as derivatization reagents, as previous studies indicated these are the most appropriate compounds for profiling of plant metabolites. The protocol is relatively rapid, delivers reproducible results, and can be employed to profile metabolites of many other types of plant tissues with only minor modifications. In this context, developing seeds can serve as an excellent system for studying metabolic regulation, since during their development, a massive synthesis of reserve compounds occurs controlled under tight transcriptional regulation and associated with temporally distinct metabolic switches.

Strigolactones (SLs) are carotenoid-derived signaling chemicals containing two lactone moieties in their structures and induce seed germination of root parasitic plants, Striga and Orobanche spp. In the rhizosphere, SLs are essential host recognition signals not only for root parasitic plants but also for arbuscular mycorrhizal fungi. In plants, SLs play important roles as plant hormones regulating shoot and root architecture. Plants produce only trace amounts of chemically unstable SLs, which makes it difficult to determine SL contents in plant tissues. Here, we describe how to extract and quantify sorgomol and 5-deoxystrigol, major SLs produced in sorghum roots.

The long-distance translocation of metabolites and mineral elements is crucial for plant growth and reproduction. In most cases, source-to-sink translocation of metabolites and minerals requires their passage through the apoplast, irrespective whether they are transported via the xylem or the phloem. This apoplast-mediated pathway is of particular importance during plant senescence, when photoassimilates as well as organic, inorganic or chelated forms of nutrients are translocated from leaves to fruits or seeds. Recent genetic and physiological studies revealed the involvement of numerous membrane transporters mediating phloem loading of amino acids, sugars, urea or mineral elements. To evaluate the contribution of individual transporters to xylem unloading or phloem loading, the collection of apoplastic fluids and of phloem sap is essential. Here, we describe a method for the extraction of apoplastic fluids and the collection of leaf petiole exudates from Arabidopsis leaves, the latter representing an approximation to the real composition of the phloem sap.

The periderm and exodermis of taproots and tuberous taproots contain an extracellular lipid polymer, suberin, deposited in their cell walls. This polymer is intractable in organic solvents, and is co-deposited with chloroform-extractable waxes. These suberin-associated root waxes are typically composed of alkanes, primary alcohols, fatty acids, alkyl ferulates, alkyl caffeates, and alkyl coumarates (Espelie et al., 1980; Li et al., 2007; Kosma et al., 2015). They are believed to contribute to the diffusion barrier properties of suberized cell walls (Soliday et al., 1979), and possibly have other roles yet to be discovered. Here we describe a protocol to extract and analyze waxes associated with root suberin. This fraction of aliphatic components is extracted by whole root immersion in chloroform, and is then chemically modified to prepare samples that are more suitable to gas-chromatography analysis. This protocol is optimized for Arabidopsis thaliana, but can be used with roots of other plants as described herein.

A plethora of natural products, mostly secondary metabolites, are isolated and purified from many different organisms, like plants, fungi, algae, marine invertebrates, etc. The extraction procedure is specific to each organism, but some guidelines are usually followed for any purification procedure regarding targeted metabolites, such as alkaloids. Alkaloids are secondary metabolites that contain basic nitrogen in their structures and they are often associated with interesting biological properties especially in pharmacology field. This protocol describes the isolation procedure of indole alkaloids from Rauvolfia nukuhivensis directly from the ethanol extract of the plant material yielding different skeleton-type compounds including non-basic derivatives (ajmaline, sarpagine, macroline and β-carboline). The procedure details the guidelines and the steps to characterize new or known isolated compounds, beginning from the plant collection to the molecule level with the use of spectroscopic techniques (NMR, MS, UV). We detailed the extraction and fractionation procedures followed by the purification of compounds, as well as their physico-chemical characterizations. The procedure is illustrated by the example of the purification of a large array of indole alkaloids from the bark of Rauvolfia nukuhivensis.

We made the method for Arabidopsis metabolome analysis based on direct-infusion Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) (IonSpec). This method was sufficiently applied to metabolic phenotyping of Arabidopsis. This method is simple in that after homogenizing samples, powdered samples are dissolved in extraction solvents (acetone and methanol) to 20% fresh weight/volume. Extracted sample solutions are dried and dissolved in 50% (v/v) acetonitrile. Mass analysis using FT-ICR/MS (IonSpec) is performed in positive and negative ionization operation modes. Mass spectra are acquired over the 100-1,000 m/z range and accumulated to improve the S/N ratio.