Chlorophyll breakdown: Pheophorbide a oxygenase is a Rieske-type iron–sulfur protein, encoded by the accelerated cell death 1 gene

Abstract

Chlorophyll (chl) breakdown during senescence is an integral part of plant development and leads to the accumulation of colorless catabolites. The loss of green pigment is due to an oxygenolytic opening of the porphyrin macrocycle of pheophorbide (pheide) a followed by a reduction to yield a fluorescent chl catabolite. This step is comprised of the interaction of two enzymes, pheide a oxygenase (PaO) and red chl catabolite reductase. PaO activity is found only during senescence, hence PaO seems to be a key regulator of chl catabolism. Whereas red chl catabolite reductase has been cloned, the nature of PaO has remained elusive. Here we report on the identification of the PaO gene of Arabidopsis thaliana (AtPaO). AtPaO is a Rieske-type iron-sulfur cluster-containing enzyme that is identical to Arabidopsis accelerated cell death 1 and homologous to lethal leaf spot 1 (LLS1) of maize. Biochemical properties of recombinant AtPaO were identical to PaO isolated from a natural source. Production of fluorescent chl catabolite-1 required ferredoxin as an electron source and both substrates, pheide a and molecular oxygen. By using a maize lls1 mutant, the in vivo function of PaO, i.e., degradation of pheide a during senescence, could be confirmed. Thus, lls1 leaves stayed green during dark incubation and accumulated pheide a that caused a light-dependent lesion mimic phenotype. Whereas proteins were degraded similarly in wild type and lls1, a chl-binding protein was selectively retained in the mutant. PaO expression correlated positively with senescence, but the enzyme appeared to be posttranslationally regulated as well.

Stay-green protein, defective in Mendel’s green cotyledon mutant, acts independent and upstream of pheophorbide a oxygenase in the chlorophyll catabolic pathway

“…3A, B). LHCII was fully retained during the course of senescence in JI2775, a feature that has been described in different stay-green mutants (Hilditch et al, 1989;Bachmann et al, 1994;Pružinská et al, 2003;Park et al, 2007), but in contrast to the stay-green mutant Bf993 of Festuca pratensis (Thomas and Howarth, 2000), proteolytic fragments of LHCII could not be detected in JI2775. In addition, core subunits of PSI (PsaD) and PSII (PsbA) were also retained to some extend in the mutant, and degradation of Rubisco subunits was somewhat slower in JI2775 compared to JI4.…”

“…Hence, the gene mutations of type C mutants were considered to affect the pathway of chl breakdown directly. This has been confirmed and is obvious for pao1 and nyc1, which are affected in catabolic steps of the pathway (Pružinská et al, 2003;Pružinská et al, 2005;Kusaba et al, 2007). The situation is different for sgr/sid mutants, since so far the function of SGR/SID could not unequivocally be demonstrated.…”

A Role for TIC55 as a Hydroxylase of Phyllobilins, the Products of Chlorophyll Breakdown during Plant Senescence

“…As a consequence, the pigment is detoxified to uncolored and photodynamically inactive products that are termed phyllobilins (Kräutler, 2014). 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 formed phyllobilins.…”

Update on the biochemistry of chlorophyll breakdown

“…However, when the photosynthetic apparatus is overexcited, for example under light stress conditions, Chl can act as a photosensitizer, that can cause cell damage and death (Apel and Hirt 2004). Likewise, defects in Chl biosynthesis and degradation result in cytotoxic effects, which are caused by the accumulation of respective photodynamic metabolic intermediates (Mochizuki et al 2010;Pružinská et al 2003). Therefore, tight regulation mechanisms, well known in the case of Chl biosynthesis Tanaka 2006, 2007), are required to prevent these toxic effects.…”