Picoliter pressure-probe-electrospray-ionization mass spectrometry for in situ monitoring single plant cell metabolite changes under stress conditions

R.; ERRA BALSELLS; KEISUKE NAKATA; HIROSHI WADA,; HIROSHI NONAMI

Florencia

Conferencia; IMSC2018; 2018

IMSF

Picoliterpressure-probe-electrospray-ionization mass spectrometry for in situ monitoringsingle plant cell metabolite changes under stress conditionsRosa Erra-Balsells1,Keisuke Nakata2, Hiroshi Wada3, Hiroshi Nonami21University of Buenos Aires, Argentina.2Ehime University, Japan.3NARO Kyushu Okinawa AgriculturalResearch Center, Japan. Keywords: pressure probe ESI,single cell, organelle, metabolomics. Introduction: Cytoplasmic streaming occurs consuming the ATPenergy formed during glycolysis, respiration and photosynthesis, movingorganelles and metabolites all around plant cells. CPP is an instrument thatcan measure the water status and several properties of intact cells (i.e.,volume)[1]. The CPP-mass spectrometer combination (picoPPESI-MS)[2,3] allows tomeasure water status, cytoplasmic streaming velocities and metabolites in cellssequentially.Methods: picoPressure Probe for water relations. Osmometer for cell osmoticpotential.Internal Electrode Capillary picoPressure Probe Electrospray Ionization(picoPPESI) as ions source, with Orbitrap mass spectrometer (Exactive Plus,Thermo Fisher Scientific Inc., MA) as MS analyzer.nanoESI- Orbitrap mass spectrometer (Exactive Plus, Thermo FisherScientific Inc., MA).Results: Plants weresubjected to water stress. Water status (turgor pressure;water potential) slightly decreased and they recovered by osmotic adjustment in12 h. The cytoplasmic streaming velocity was maintained during osmoticadjustment. PicoPPESI-MS indicated that several phosphate-metabolites (m/z 78.96 [HPO3-],96.97 [H2PO4-], and 259.02 [Glu-6-phosphate])were transiently accumulated in the cells in response to the treatment.Furthermore, not onlymetabolites yielded by glycolysis occurred in cytosol (m/z 87.01 [Pyruvic ac.], 179.06 [Hex], 259.02 [Glu-6-phosphate],and 341.11 [Hex2]) but also those from Krebs cycle in themitochondrion (m/z 115.00 [Fumaricac.]), 117.02 [Succinic ac.), 133.01 [Malic ac.], 145.02 [á-Ketoglutaric ac.],173.01 [Aconitic ac.] and 191.02 [Citric ac.]) accumulated in response to thestress treatment. Because these metabolic pathways are essential for ATPhydrolysis, ADP production and the osmotic adjustment, is obvious theircontribution to maintain cytoplasmic streaming providing energy.Conclusions: Tacking into account the metabolic pathwaysmentioned above and the results obtained by picoPPESI-MS monitoring the cell metabolites,ATP hydrolysis, ADP production and the osmotic cell adjustment are essentialfor keeping cytoplasmic streaming providing the nececssary energy.Novel Aspect: PicoPPESI mass spectrometry system has apotentiality to monitor metabolic changes insitu at cell level as well as at organelle level. References1. Boyer J.S., Measuring the Water Status of Plants andSoils, Academic Press, San Diego (1995).2. Gholipour Y., Erra-Balsells R., Nonami H., Anal.Biochem., 433, 70 (2013).3. Nakashima T., Erra-Balsells R., Nonami H., Anal. Chem.,88, 3049 (2016).