In situ picolitre pressure-probe-electrospray-ionization mass spectrometry for monitoring single cell metabolites change.

K. NAKATA, ; H. WADA, ; Y. ONDA, ; R.; ERRA BALSELLS; H. NONAMI

Osaka

Conferencia; Mass Spectrometry and Proteomics 2018 (MSP2018); 2018

Japan Society Mass Spectrometry

Insitu picolitre pressure-probe-electrospray-ionizationmass spectrometry for monitoring single cell metabolites change (Ehime University1, NAROKyushu Okinawa Agricultural Research Center2, University of BuenosAires3) ??Keisuke Nakata1,Hiroshi Wada2, Yayoi Onda1, Rosa Erra-Balsells3,Hiroshi Nonami1 Single cellmetabolomics, called picolitre pressure-probe-electrospray-ionization massspectrometry (picoPPESI-MS), was used to directly examine the relationshipbetween cytoplasmic streaming, cell metabolites, and cell water relations in intacttomato (Solanum lycopersicum L. ?eMicro-Tom?f)trichome cells. Cytoplasmic streaming was monitored under the digital microscope.And, cell metabolites were determined by picoPPESI-MS immediately after cellularfluids were collected from the cells using a cell pressure-probe (CPP). When theplants were subjected to water stress, the water potential and turgor pressure bothdeclined; however, the cytoplasmic streaming velocity was maintained throughouttreatment. Metabolites in glycolysis in cytosol and Krebs cycle in mitochondriawere accumulated significantly in response to the water stress for osmoticadjustment, which sustained cytoplasmic streaming under the stress conditions. Becausethe system allowed us to identify the source organelles of the metabolites inthe collected cell fluids, we conclude that thisanalytical method is capable of sequentially determining organelle-specific metabolicchanges in intact plant cells under a given stress condition. Keywords: pressure probe electrospray, single cell analysis, organellestress response Cytoplasmicstreaming occurs as myosin-linked organelles that move along actin filaments byconsuming the energy of adenosine triphosphate (ATP) formed during glycolysis,respiration, and photosynthesis to carry organelles and molecules, such asnutrients and metabolites, to all parts of the cell. In this work, we usedstalk cells of type II trichomes in tomato plants as a model to study howcytoplasmic streaming and cell metabolites change when the plant water status wasaltered. CPP is an instrument that can measure the water status and severalproperties of intact single cells including cell volume determination1).Combining the CPP and an orbitrap mass spectrometer, the picoPPESI-MS2),3) allows to measure the water status, cytoplasmic streaming velocitiesand concentrations of metabolites in plant cells sequentially. After the plants were subjected to moderate waterstress, the water status (turgor pressure and water potential) slightlydecreased after the initiation of the treatment, and they recovered by osmoticadjustment in 12 h. The cytoplasmic streaming velocity was found to bemaintained during osmotic adjustment. PicoPPESI-MS analysis also indicated thatseveral phosphate-metabolites (m/z78.96 [HPO3-], 96.97 [H2PO4-],and 259.02 [Glucose-6-phosphate]) were transiently accumulated in the cells inresponse to the treatment. Furthermore, notonly metabolites of glycolysis occurred in cytosol (m/z 87.01 [Pyruvic acid], 179.06 [Hex], 259.02 [Glucose-6-phosphate],and 341.11 [Hex2]) but also metabolites of Krebs cycle in the matrixof the mitochondrion (m/z 115.00 [Fumaricacid]), 117.02 [Succinic acid), 133.01 [Malic acid], 145.02 [?¿-Ketoglutaricacid], 173.01 [Aconitic acid] and 191.02 [Citric acid]) were accumulated oractivated in response to the treatment. Because these metabolic pathways are essentialfor ATP hydrolysis, ATP production, and the osmotic adjustment, the results highlighttheir contribution to maintain cytoplasmic streaming by providing energy. Therefore,we conclude that the system has a potential to monitor metabolic changes in situ at organelle level, rather thancell level.