The PLUMED consortium (2019). Promoting transparency and reproducibility in enhanced molecular simulations. Nature Methods, 16(8), 670-673. https://doi.

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.

During senescence, chlorophyll (chl) is metabolized to colorless nonfluorescent chl catabolites (NCCs). A central reaction of the breakdown pathway is the ring cleavage of pheophorbide (pheide) a to a primary fluorescent chl catabolite. Two enzymes catalyze this reaction, pheide a oxygenase (PAO) and red chl catabolite reductase. Five NCCs and three fluorescent chl catabolites (FCCs) accumulated during dark-induced chl breakdown in Arabidopsis (Arabidopsis thaliana). Three of these NCCs and one FCC (primary fluorescent chl catabolite-1) were identical to known catabolites from canola (Brassica napus). The presence in Arabidopsis of two modified FCCs supports the hypothesis that modifications, as present in NCCs, occur at the level of FCC. Chl degradation in Arabidopsis correlated with the accumulation of FCCs and NCCs, as well as with an increase in PAO activity. This increase was due to an up-regulation of Pao gene expression. In contrast, red chl catabolite reductase is not regulated during leaf development and senescence. A pao1 knockout mutant was identified and analyzed. The mutant showed an age-and light-dependent cell death phenotype on leaves and in flowers caused by the accumulation of photoreactive pheide a. In the dark, pao1 exhibited a stay-green phenotype. The key role of PAO in chl breakdown is discussed.Chlorophyll (chl) degradation is an integral part of leaf senescence and fruit ripening. The fate of chl during senescence has been well established in recent years (for review, see Matile et al., 1999; Hörtensteiner, 1999; Hö rtensteiner and Kräutler, 2000;Kräutler, 2003;Eckhardt et al., 2004). Thereby, chl is converted to colorless nonfluorescent chl catabolites (NCCs; Fig. 1) in a pathway that is probably active in all higher plants (Pružinská et al., 2003;Gray et al., 2004). Structure elucidation of NCCs from different species has unraveled a common tetrapyrrolic skeleton with an oxygenolytically opened porphyrin macrocycle (Kräutler, 2003). Peripheral modifications at several side chains within different NCCs (Fig. 1, R 1 -R 3 ) are species specific (Berghold et al., 2002(Berghold et al., , 2004, and hence have been proposed to occur rather late in the pathway (Hö rtensteiner, 1999). Indeed, a primary chl breakdown product (primary fluorescent chl catabolite-1 [pFCC-1]), which exhibits a blue fluorescence, could be identified as a common product of porphyrin ring cleavage ( Fig. 1; Mü hlecker et al., 1997). Thus, the sequence of reactions is the removal of phytol and magnesium (Mg) by chlorophyllase and Mg-dechelatase, respectively, followed by the conversion of pheophorbide (pheide) a to pFCC-1, which requires the activity of two enzymes, pheide a oxygenase (PAO) and red chl catabolite (RCC) reductase (RCCR; Rodoni et al., 1997;Hö rtensteiner, 1999).PAO is a chloroplast envelope-bound Rieske-type iron-sulfur oxygenase, which is identical to lethal leaf spot 1 (LLS1) from maize (Zea mays) and accelerated cell death 1 (ACD1) from Arabidopsis (Arabidopsis thaliana; Pružinská et al., 2003;Yang e...

A central reaction of chlorophyll breakdown, porphyrin ring opening of pheophorbide a to the primary fluorescent chlorophyll catabolite (pFCC), requires pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR), with red chlorophyll catabolite (RCC) as a presumably PAO-bound intermediate. In subsequent steps, pFCC is converted to different fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs). Here, we show that RCCR-deficient Arabidopsis thaliana accumulates RCC and three RCC-like pigments during senescence, as well as FCCs and NCCs. We also show that the stereospecificity of Arabidopsis RCCR is defined by a small protein domain and can be reversed by a single Phe-to-Val exchange. Exploiting this feature, we prove the in vivo participation of RCCR in chlorophyll breakdown. After complementation of RCCR mutants with RCCRs exhibiting alternative specificities, patterns of chlorophyll catabolites followed the specificity of complementing RCCRs. Light-dependent leaf cell death observed in different RCCRdeficient lines strictly correlated with the accumulation of RCCs and the release of singlet oxygen, and PAO induction preceded lesion formation. These findings suggest that RCCR absence causes leaf cell death as a result of the accumulation of photodynamic RCC. We conclude that RCCR (together with PAO) is required for the detoxification of chlorophyll catabolites and discuss the biochemical role(s) for this enzyme.

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