Abstract
Chlorophyll degradation is the most obvious hallmark of leaf senescence. Phyllobilins, linear tetrapyrroles that are derived from opening of the chlorin macrocycle by the Rieske-type oxygenase PHEOPHORBIDE a OXYGENASE (PAO), are the end products of chlorophyll degradation. Phyllobilins carry defined modifications at several peripheral positions within the tetrapyrrole backbone. While most of these modifications are species-specific, hydroxylation at the C32 position is commonly found in all species analyzed to date. We demonstrate that this hydroxylation occurs in senescent chloroplasts of Arabidopsis thaliana. Using bell pepper (Capsicum annuum) chromoplasts, we establish that phyllobilin hydroxylation is catalyzed by a membrane-bound, molecular oxygen-dependent, and ferredoxin-dependent activity. As these features resemble the requirements of PAO, we considered membrane-bound Rieske-type oxygenases as potential candidates. Analysis of mutants of the two Arabidopsis Rieske-type oxygenases (besides PAO) uncovered that phyllobilin hydroxylation depends on TRANSLOCON AT THE INNER CHLOROPLAST ENVELOPE55 (TIC55). Our work demonstrates a catalytic activity for TIC55, which in the past has been considered as a redox sensor of protein import into plastids. Given the wide evolutionary distribution of both PAO and TIC55, we consider that chlorophyll degradation likely coevolved with land plants.
Highlights
Chlorophyll is the main pigment of the photosynthetic apparatus of plants and is responsible for the absorption of the energy of sunlight
If any, chlorophyll catabolites are found in Arabidopsis plastids, isolated gerontoplasts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
When incubating at pH 5, epi-primary fluorescent chlorophyll catabolite (pFCC) isomerized to a more polar compound (Supplemental Figure 1B), which when analyzed by tandem MS fragmented as a typical NCC (Christ et al, 2016), i.e., with high probability of ring D loss (Supplemental Figure 1A), confirming the identity of the catabolites isolated from senescent Arabidopsis chloroplasts as FCCs, as pFCC and hydroxy-pFCC, which solely differ by the attachment in the latter of an additional oxygen atom at ring A, likely at the C32 ethyl side chain
Summary
Chlorophyll is the main pigment of the photosynthetic apparatus of plants and is responsible for the absorption of the energy of sunlight. Chlorophyll degradation to phyllobilins is catalyzed by a multistep process, named the “PAO/phyllobilin” pathway (Hörtensteiner and Kräutler, 2011; Kräutler and Hörtensteiner, 2014), to acknowledge PAO (PHEOPHORBIDE a OXYGENASE), a Rieske-type oxygenase that is the key component of this pathway (Pruzinská et al, 2003). PAO, in concert with red chlorophyll catabolite reductase (RCCR), catalyzes the ring-opening reaction of pheophorbide a, the phytol- and Mg-free intermediate of the early part of chlorophyll breakdown, to a primary fluorescent chlorophyll catabolite (pFCC), the primary (fluorescent) phyllobilin that is the precursor of all subsequently
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