Characterization of the pheophorbide a oxygenase/phyllobilin pathway of chlorophyll breakdown in grasses

Abstract

Although the PAO/phyllobilin pathway of chlorophyll breakdown is active in grass leaf senescence, the abundance of phyllobilins is far below the amount of degraded chlorophyll. The yellowing of fully developed leaves is the most prominent visual symptom of plant senescence. Thereby, chlorophyll is degraded via the so-called pheophorbide a oxygenase (PAO)/phyllobilin pathway to a species-specific set of phyllobilins, linear tetrapyrrolic products of chlorophyll breakdown. Here, we investigated the diversity and abundance of phyllobilins in cereal and forage crops, i.e. barley, rice, ryegrass, sorghum and wheat, using liquid chromatography-mass spectrometry. A total of thirteen phyllobilins were identified, among them four novel, not yet described ones, pointing to a rather high diversity of phyllobilin-modifying activities present in the Gramineae. Along with these phyllobilins, barley orthologs of known Arabidopsis thaliana chlorophyll catabolic enzymes were demonstrated to localize in the chloroplast, and two of them, i.e. PAO and pheophytin pheophorbide hydrolase, complemented respective Arabidopsis mutants. These data confirm functionality of the PAO/phyllobilin pathway in grasses. Interestingly, when comparing phyllobilin abundance with amounts of degraded chlorophyll in senescent leaves, in most analyzed grass species only minor fractions of chlorophyll were recovered as phyllobilins, opposite to A. thaliana where phyllobilin quantities match degraded chlorophyll rather well. These data show that, despite the presence and activity of the PAO/phyllobilin pathway in barley (and other cereals), phyllobilins do not accumulate stoichiometrically, implying possible degradation of chlorophyll beyond the phyllobilin level.

Chlorophyll breakdown—Regulation, biochemistry and phyllobilins as its products

“…The observation is intriguing that abundance of PBs is low in senescent leaf tissue in many plant species despite the obvious visible signs of Chl breakdown (Das et al, 2018). This raises interesting questions regarding the final fate of Chl during breakdown, which 30 years ago was still enigmatic in many respects (Matile et al, 1987).…”

“…CYP89A9 localizes to the ER, and oxidatively deformylates FCCs, but not NCCs. The occurrence of DNCCs and NCCs varies among different plant species (Das, Christ, & Hörtensteiner, 2018). In contrast to Arabidopsis, which accumulates both types of PBs, in C. japonicum exclusively NCCs are found Oberhuber et al, 2003), while in Norway maple one single DNCC accumulates, but no NCCs (Müller et al, 2011).…”

“…C18-dihydroxylated PBs have been identified in many plants; in particular, they have been found in all grass species analysed to date (Berghold, Müller, Ulrich, Hörtensteiner, & Kräutler, 2006;Das et al, 2018;Kräutler et al, 1991), while they are absent from several families of angiosperms such as the Brassicaceae (Christ et al, 2016;Mühlecker & Kräutler, 1996;Pružinská et al, 2005). This indicates the presence of a respective dihydroxylating activity in only certain plant clades.…”

“…This indicates the presence of a respective dihydroxylating activity in only certain plant clades. 18 O2-labeling experiments showed that both oxygen atoms incorporated into the C18-dihydroxyethyl group are derived from molecular oxygen, indicating that the responsible enzyme(s) could be a dioxygenase (Das et al, 2018). However, the molecular identity of such a dioxygenase remains elusive.…”

“…Whether the known PBs represent the end stage of Chl breakdown or if degradation occurs beyond them in viable leaf tissue is not clear yet. It has been reported that the amount of degraded Chl does not always correlate with the amount of accumulated PBs (Berghold et al, 2002;Christ et al, 2016;Das et al, 2018;Kräutler et al, 1992;Losey & Engel, 2001). Catabolite analysis of leaves of a Nicotiana benthamiana plant that were selected along the stem, i.e.…”