Mass spectrometry: the indispensable tool for plant metabolomics of colourless chlorophyll catabolites

Senescence and ripening of plant tissues engage the pheophorbide a oxygenase pathway, reducing the chlorophyll content to inactive chlorophyll catabolite products, termed phyllobilins. These products are open-macrocycle derivatives, but present different structural features related to species-dependent enzyme activity. This review encompasses a brief outline of the chlorophyll catabolism pathway, a detailed description of the structural motifs of known phyllobilins, giving details of how mass spectrometry provides hints for the characterization of phyllobilins. The structural approach for the identification of phyllobilins requires several spectroscopic methodologies to reach a complete structural identification, including UV–visible spectroscopy, circular dichroism, nuclear magnetic resonance and mass spectrometry. Among these techniques, mass spectrometry presents several advantages for showing the structural features of phyllobilins, through acquisition of accurate mass, elemental composition, and detection of product ions, which provide valuable structural information. The combination of mass spectra with data-managing and in silico prediction tools greatly enhances the comprehensive building of the phyllobilin structure, and the resolving of the intriguing puzzle of enzymatic and chemical reactions involved in chlorophyll catabolism. Indeed, some strategies based on structural constraints that phyllobilins present, with recent developments in software prediction tools are proposed to foster the unravelling of phyllobilin structures.