Blossom End Rot Tomato Fruit Diagnosis for In Situ Cell Analyses with Real Time Pico-Pressure Probe Ionization Mass Spectrometry

Blossom end rot (BER) of tomato fruit has been identified as a physiological disorder caused by calcium (Ca) deficiency (Lyon et al., 1942). Ca has been believed to adjust the structure and properties, particularly gelation, of cell wall pectin (Jarvis, 1984). Gelation, which directly influences the elasticity and expansion of cell wall, is critically dependent on the availability of Ca in the apoplast. It has been speculated that the insufficient availability of Ca in the apoplast of expanding cells results in cell wall weakening, impairment of cell expansion, and finally cell burst and death (Ho and White, 2005). However, high Ca concentration could induce BER in tomato fruit (Nonami et al., 1995; Hossain and Nonami, 2012). Ca concentration in BER fruit was higher than that in healthy fruit (Nonami et al., 1995). Thus, it may be possible to speculate that some factors other than Ca deficiency causes BER in tomato fruit. When Ca salts were added excessively to the hydroponic solution, we could induce BER in 30 50% of fruit formed in tomato plants (Nonami et al., 1995; Hossain and Nonami, 2012). It is noteworthy that 50 70% of fruit in the same tomato plant did not exhibit BER although the fruit was exposed to the same stress condition. All cells in both BER and non-BER exhibiting fruit in the same plant had the same DNA, having different metabolisms. How was this difference induced? Analysis of cell physical and chemical properties with single cell resolution can clarify cell to cell variations and many primary growth, disorder, or stress related phenomena that cannot be detected or fully explored through tissue-level studies. Integrative analyses of water relations and metabolomics of plant cells, therefore, can provide remarkable insights to many physiological events during growth or environmental stresses. However common water status measurements are not provided with molecular information and on other hand, a big challenge is to perform quantitative metabolite profiling at cell level. In order to investigate these distinct aspects concurrently at real time and with single cell resolution, we devised a new technique by combining a cell pressure probe (PP) and an Orbitrap mass spectrometer, named as pico-pressure probe ionization mass spectrometry (picoPPI-MS). The PP is routinely used to analyze several properties of plant single cells including in situ cell volume determination (Malone and Tomos, 1990), and turgor pressure, osmotic potential, water potential, plasma membrane hydraulic conductivity, and cell wall elastic modulus measurements (Nonami and Boyer, 1989; Nonami and Schulze, 1989; Boyer, 1995). In addition PP uniquely facilitated managed picoliter sampling of in situ single cell solution, since sample volume can be controlled and measured (Nonami and Schulze, 1989). In picoPPI-MS, after the