Pico pressure probe electrospray mass spectrometry for in situ single plant cell qualitative and quantitative analysis of phosphate metabolic pathways.

K. NAKATA, ; WADA, HIROSHI; R.; ERRA BALSELLS; H. NONAMI

Osaka

Conferencia; The Mass Spectrometry Society of Japan, The 65th Annual Conference on Mass Spectrometry; 2017

The Mass Spectrometry Society of Japan

Pico pressure probe electrospraymass spectrometry for in situ singleplant cell qualitative and quantitative analysis of phosphate metabolicpathways(Ehime University1, NAROKyushu Okinawa Agricultural Research Center2, University of BuenosAires3) ¡ðKeisuke Nakata1,Hiroshi Wada2, Rosa Erra-Balsells3, Hiroshi Nonami1Shortabstract: Cytoplasmic streaming occurs as myosin-linkedorganelles move along actin filaments by using the energy of adenosine triphosphate (ATP) formed during glycolysis, respiration andphotosynthesis to carry organelles, nutrients, metabolites, geneticinformation, and other materials to all parts of the cell. By combining the cell pressureprobe and an orbitrap mass spectrometer, cell turgor (¦·p), osmoticpotential (¦·s), water potential (¦·w), cytoplasmicstreaming velocity, and metabolites of stalk cells of type II trichomes on theadaxial sixth leaf surface of tomato (Solanumlycopersicum L. cv. Micro-Tom) in a growth chamber were measured before andafter water stress treatment. Furthermore, we evaluated phosphate metabolicpathways of the single cell qualitatively and quantitively.Keywords: pressure probe electrospray, single cell quantitativeanalysis, dynamic range, limit of detection Fig. 3. Chemical structural formulas of ATP (A) and G6P (B).    A cell pressure probe is an instrument thatcan measure the water status and several properties of intact single cellsincluding cell volume determination. The position of the meniscus between thecell sap and the oil in the microcapillary is controlled by moving the metalrod1). Combining the cell pressure probe and an orbitrap massspectrometer, the pico pressure probe electrospray mass spectrometry (PPPESI-MS)system can be performed high spatial-resolution cell sampling, precise volumecontrol, and high detection sensitivity2). After the plants weresubjected to the water stress, the water status (¦·p, ¦·s,and ¦·w) of the stalk cells declined immediately,and stabilized in 30 minutes. Complete recovery of the water status wasobserved after 12 h of the treatment. By contrast, the cytoplasmic streamingvelocity was maintained during the treatment, and some phosphate signalintensities increased immediately after the treatment (Fig. 1.). In this study,the method to quantitively evaluate glycolysis and Krebs cycle of a living singlecell was provided by measuring some phosphate salts standard solutions with thesame PPPESI-MS system condition. The peaks of [HPO3]- and[H2PO4]- increased immediately after thetreatment indicated the relation between the signal intensities or chromatographicarea and number of moles had linear relationships between 200 attomoles and 100femtomoles (Fig. 2.). Furthermore, the fragment ion of [HPO3]-or [H2PO4]- was detected as high signalintensity from each ATP and glucose-6-phosphate (G6P) with the measurementsystem (Fig. 3). Therefore, the method could evaluate glycolysis and Krebscycle of the single cell quantitively.               Fig. 1. PPPESI negative ion mode mass spectra obtained from a stalk cell of type II trichome on the adaxial sixth leaf surface before, immediately after, and 12 hours after the 1X modified Hoagland solution treatments (A, B, and C respectively).     Fig. 2. Signal intensity of [HPO3]- (m/z 78.96) (A) and [H2PO4]- (m/z 96.97) (B) vs number of moles. PPPESI negative ion mode; NaH2PO4 concentration rage of 10 n - 500 ¦ÌM, 200 pL injection sample volume. The values are mean ¡À SE (n = 3 - 9).           ReferencesBoyer, J. S., Measuringthe Water Status of Plants and Soils, Academic Press, San Diego, 1-495 (1995).Nakashima, T. etal., Anal. Chem., 88, 3049¨C3057(2016).