Single-cell metabolite profiling of stalk and glandular cells of intact trichomes with internal electrode capillary pressure probe electrospray ionization mass spectrometry.

H. NONAMI, ; T. NAKASHIMA, ; H. WADA, ; S. MORITA, ; K. HIRAOKA,; ROSA ERRA-BALSELLS; R, ERRA BASELLS,

Osaka

Conferencia; The 64th Annual Conference on Mass Spectrometry, Osaka,; 2016

The Mass Spectrometry Society of Japan,

 Keywords: Pressure probe, Single-cell metabolomics, Tandem MS, Tomatotrichomes  Plant trichomes areunicellular or multicellular epidermal outgrowths displaying various morphologies,functions, and distribution patterns across organs. For this reason, they are consideredas an ideal model cellular system to study mechanisms underlying cellulardifferentiation, interaction and function at single cell resolution. In Solanum spp., eight distinct types of trichomeshave been described and classified into glandular or non-glandular types,depending on the presence or absence of glandular cells atop the stalk cells.1Since the metabolic ability of glandular cells to synthesize and accumulatemetabolites of potential importance for pharmaceutical industries andanti-herbivory defenses of tomato plants were appreciated, extensive omicsstudies have been conducted on isolated glandular cells.2,3 However, metabolite analyseson stalk cells have been completely lacking thus far. This is largely due todifficulties in selective sampling or isolation of stalk cells withoutcontamination of adjacent glandular or epidermal cells. Toward a deeperunderstanding of metabolism in glandular trichomes, clarifying metabolic variabilityand interaction between the two cell types is crucial. Here, we report singlecell sampling and subsequent MS analysis of both stalk and glandular cells intype I, IV and VI trichomes on tomato plants with the aid of internal electrodecapillary pressure probe electrospray ionization (IEC-PPESI-MS) technique.4Upon the measurement, a taperedquartz microcapillary filled with 0.01% (v/v) trihexyl (tetradecyl) phosphoniumbis trifluoromethanesulfonyl amide in silicone oil was fitted airtightly to acapillary holder in which titanium wire was pre-embedded, so that the internalwire electrode was inserted into the microcapillary through the rear openingand settled near the tip end. The microcapillary tip was first impaled intotargeted stalk cells for sap sampling, then pulled out of the cell and finally orientedtowards the orifice of an orbitrap mass spectrometer (Exactive Plus, ThermoFisher Scientific Inc., MA) at approximately 3 mm distance. Subsequently, the sampletrapped inside the tip was electrified with -4 kV via the internal electrodeconnected to a high voltage generator (AKTB-05k1PN/S, Touwa Keisoku Corp.,Tokyo, Japan) for ionization and detection of the metabolites. For the analysisof glandular cells, on the other hand, the glandular cell component was sampled on the external wall of thetip with gentle contact because of its high viscosity, which hindered directsampling of cell sap into the microcapillary and electrospray ionizationunfeasible. In this case, the tip holding sample was briefly dipped into a 500nL droplet of ultrapure water formed at a micropipette tip, and at the sametime, approximately 300 pL of the solution was loaded into the microcapillary. Using this sampling method, highly viscoussample was instantly and locally dissolved into the water droplet, and thediluted sample was simultaneously trapped into the microcapillary. After the dilutionprocedure, negative ion mode measurements were performed. In addition, flavonoidsand acyl sugars were identified by tandem MS coupled with IEC-PPESI.A number of peaks of aminoacids, organic acids, carbohydrates and flavonoids and acyl sugars weredetected from single stalk and/or glandular cells with a minimum of sub-picolitersap sample. The minimal cell sap removal from a stalk cell without severedisturbance of trichome structure also allowed sequential analysis of adjacentglandular cell on the same type VI trichome, which showed the presence ofstriking difference in metabolite compositions between two adjacent cell types.Some metabolites were found only in specific cell types or particular trichometypes. Cell-to-cell differences in metabolite compositions between stalk cellsand glandular cells in the same trichome type as well as among differenttrichome types will be further discussed. ReferencesJ. Glas et al., Int. J. Mol. Sci. 13 17077-17103 (2012)D. Tian et al., Planta 236 1053-1066 (2012)A. Schilmiller et al., Plant J. 54 702-711 (2008)T. Nakashima et al., Anal. Chem. In press (2016)