Combination of electronically driven micromanipulation with laser desorption ionization mass spectrometry - The unique tool for analysis of seed coat layers and revealing the mystery of seed dormancy.

Electronically driven micromanipulation (EDM) with microscopic control was used as a novel tool for sample preparation prior to direct (matrix assisted) laser desorption/ionization mass spectrometric ((MA)LDI-MS) analysis of mature pea seed coat composition in defined layers. Microscissors were used for seed coat fragment shape adjustment, microtweezers for sample holding and "microjackhammer" Milling Pro for precise mechanical removing of cell layers in defined depths (2, 5 or 10 μm). These procedures circumvent the application of embedding media or enzymatic digestion of seed coat that would complicate mass spectra interpretation (presence of matrix signals, analyte signals enhancement or attenuation) and represent alternative for 3D metabolites profiling. In addition, microinjector was used to apply a solution on intact or micropeeled seed coat surface in nano-volumes, i.e. MALDI matrix and/or lithium salt, that provide improvement of signal of sugars. Utilization of EDM enabled optimization of matrix composition on a single small fragment of seed coat overcoming thus problems with biological (seed to seed) variability. LDI-MS data were studied by multivariate statistical analysis and significant metabolites in particular layers of seed coats were identified. Normalized intensities of signals (NS) of long-chain hydroxylated fatty acids (HLFA) on intact dormant pea genotype (JI64) seed coats were significantly higher than in their counterparts treated by micropeeling confirming HLFA accumulation in outermost layers (cutin). Fatty acids distribution differences between dormant and non-dormant genotypes were explored in detail. On the other hand, NS of sugar chains and particular polyphenols were significantly higher in micropeeled seed coats of studied dormant and non-dormant genotypes than in intact seed coats. Furthermore, combination of EDM with mass spectrometry imaging (MSI) allowed vertical profiling of metabolites in hilum (a place of former attachment of seed to maternal plant) and comparison of its composition with surrounding tissues. The obtained results contribute to the understanding of relations between seed coat chemical composition and physical seed dormancy.