SUMMARYWe discuss and illustrate recent advances that have been made to image the distribution of metabolites among cells and tissues of plants using different mass spectrometry technologies. These technologies include matrixassisted laser desorption ionization, desorption electrospray ionization, and secondary ion mass spectrometry. These are relatively new technological applications of mass spectrometry and they are providing highly spatially resolved data concerning the cellular distribution of metabolites. We discuss the advantages and limitations of each of these mass spectrometric methods, and provide a description of the technical barriers that are currently limiting the technology to the level of single-cell resolution. However, we anticipate that advances in the next few years will increase the resolving power of the technology to provide unprecedented data on the distribution of metabolites at the subcellular level, which will increase our ability to decipher new knowledge concerning the spatial organization of metabolic processes in plants.

High-spatial resolution and high-mass resolution techniques are developed and adopted for the mass spectrometric imaging of epicuticular lipids on the surface of Arabidopsis thaliana. Single cell level spatial resolution of approximately 12 mum was achieved by reducing the laser beam size by using an optical fiber with 25 mum core diameter in a vacuum matrix-assisted laser desorption ionization-linear ion trap (vMALDI-LTQ) mass spectrometer and improved matrix application using an oscillating capillary nebulizer. Fine chemical images of a whole flower were visualized in this high spatial resolution showing substructure of an anther and single pollen grains at the stigma and anthers. The LTQ-Orbitrap with a MALDI ion source was adopted to achieve MS imaging in high mass resolution. Specifically, isobaric silver ion adducts of C29 alkane (m/z 515.3741) and C28 aldehyde (m/z 515.3377), indistinguishable in low-resolution LTQ, can now be clearly distinguished and their chemical images could be separately constructed. In the application to roots, the high spatial resolution allowed molecular MS imaging of secondary roots and the high mass resolution allowed direct identification of lipid metabolites on root surfaces.

The ontogeny of seed structure and the accumulation of seed storage substances is the result of a determinant genetic program. Using RNA interference, the synthesis of soybean (Glycine max) glycinin and conglycinin storage proteins has been suppressed. The storage protein knockdown (SP2) seeds are overtly identical to the wild type, maturing to similar size and weight, and in developmental ontogeny. The SP2 seeds rebalance the proteome, maintaining wild-type levels of protein and storage triglycerides. The SP2 soybeans were evaluated with systems biology techniques of proteomics, metabolomics, and transcriptomics using both microarray and next-generation sequencing transcript sequencing (RNA-Seq). Proteomic analysis shows that rebalancing of protein content largely results from the selective increase in the accumulation of only a few proteins. The rebalancing of protein composition occurs with small alterations to the seed's transcriptome and metabolome. The selectivity of the rebalancing was further tested by introgressing into the SP2 line a green fluorescent protein (GFP) glycinin allele mimic and quantifying the resulting accumulation of GFP. The GFP accumulation was similar to the parental GFPexpressing line, showing that the GFP glycinin gene mimic does not participate in proteome rebalancing. The results show that soybeans make large adjustments to the proteome during seed filling and compensate for the shortage of major proteins with the increased selective accumulation of other proteins that maintains a normal protein content.