Comparative metabolite profiling of intact tomato trichomes at single-cell resolution using pressure probe electrospray ionization mass spectrometry

T. NAKASHIMA, ; J. KASUGA, ; H. WADA, ; S. MORITA, ; K. HIRAOKA,; Y. GHOLIPOUR, ; H. NONAMI; ERRA-BALSELLS, R.; EDUARDO M. RUSTOY, YASUTO SATO, HIROSHI NONAMI, ROSA ERRA BALSELLS AND ALICIA BALDESSARI

Osaka

Conferencia; The 62nd Annual Conference on Mass Spectrometry; 2014

The Mass Spectrometry Society of Japan

Comparative metabolite profiling of intact tomato trichomes at single-cell resolution using pressure probe electrospray ionization mass spectrometry (Ehime University1, Kyushu Okinawa Agricultural Research Center2, University of Buenos Aires3, Yamanashi University4) ??T. Nakashima1, J. Kasuga1, H. Wada2, S. Morita2, R. Erra-Balsells3, K. Hiraoka4, Y. Gholipour1, H. Nonami1   Keywords: Electrospray ionization, Pressure probe, Single-cell metabolomics, Tomato, Trichomes, Trichomes are unicellular or multicellular outgrowths originating from the aerial epidermal cells of stems, leaves, floral organs and fruits. In Solanum spp., they are typically classified into eight distinct types based on their morphological appearance1). Some types of trichomes are known to synthesize and accumulate specialized metabolites such as acylsugars2) and terpenoids3), the content of which may vary transiently through developmental stages or depending on the growth conditions of plants. Monitoring of the physicochemical and metabolic properties of individual trichome cells is, therefore, a potential means to provide diagnostic clues for physiological status of plants. For more reliable mass spectrometry-based single-cell analysis, a precise cell sap sampling mechanism and high sensitivity in the ionization/detection system are critical prerequisites. In this regard, pressure probe electrospray ionization mass spectrometry (PPESI-MS) which involves sequential assessments of water status and metabolic profiles of single plant cells in a less destructive manner4) is considered to be a promising technique. In PPESI-MS system, the use of a finely tapered quartz capillary mounted on a X-Y-Z piezo manipulator enables highly accurate control of the tip location as well as handling and determination of sample volumes in sub-picoliter order5). Moreover, the lower limit of detection for several plant metabolites were reported to be fairly small4), and it was further improved by a series of modifications during the course of present study. By using this system, here we demonstrated metabolite profiling of intact single cells from different types of trichome on the stems, leaves, and fruits of tomato plants (Solanum lycopersicum L. cv. Micro-tom). The PPESI-MS measurement was made on stalk cells that all types of trichome possess in common (Fig. 1). A quartz capillary filled with the mixture of an ionic liquid (trihexyl (tetradecyl) phosphonium bis (trifluoromethanesulfonyl)) and silicone oil was used for the measurement. Once the capillary tip penetrated into target stalk cells, turgor pressure (Yp) was measured. Subsequently, the tip containing cell sap was pulled out of the cell and oriented towards the orifice of Orbitrap mass spectrometer (Exactive Plus, Thermo Fisher Scientific Inc. US) at 3 mm distance. The high voltage (+/- 4 kV ) was then applied to the titanium wire inserted in the capillary to initiate ionization. Mass spectra were successfully obtained with cell sap volume of 1 pL at minimum. Several major peaks of metabolite such as carbohydrate, amino acid and organic acid were observed in negative and positive ion mode mass spectra. The metabolites most frequently observed were malic acid and disaccharide in negative and positive ion mode, respectively. However, the relative abundance and number of metabolite varied among different types of trichome. Comparisons were also made between PPESI-MS mass spectra of single cells and nanoESI-MS with diluted cell sap samples to confirm the result. The type specific variations in metabolite profiles and improvements made in the PPESI-MS system will be further discussed.