Family Report for: Pheophytinase

Recent identification of NYE1/SGR1 brought up a new era for the exploration of the regulatory mechanism of Chlorophyll (Chl) degradation. Cluster analysis of senescence associated genes with putative chloroplast targeting sequences revealed several genes sharing a similar expression pattern with NYE1. Further characterization of available T-DNA insertion lines led to the discovery of a novel stay-green gene CRN1 (Co-regulated with NYE1). Chl breakdown was significantly restrained in crn1-1 under diversified senescence scenarios, which is comparable with that in acd1-20, but much more severe than that in nye1-1. Notably, various Chl binding proteins, especially trimeric LHCP II, were markedly retained in crn1-1 four days after dark-treatment, possibly due to a lesion in disassociation of protein-pigment complex. Nevertheless, the photochemical efficiency of PSII in crn1-1 declined, even more rapidly, two days after dark-treatment, compared to those in Col-0 and nye1-1. Our results suggest that CRN1 plays a crucial role in Chl degradation, and that loss of its function produces various side-effects, including those on the breakdown of Ch-protein complex and the maintenance of the residual photosynthetic capability during leaf senescence.

During leaf senescence, chlorophyll is removed from thylakoid membranes and converted in a multistep pathway to colorless breakdown products that are stored in vacuoles. Dephytylation, an early step of this pathway, increases water solubility of the breakdown products. It is widely accepted that chlorophyll is converted into pheophorbide via chlorophyllide. However, chlorophyllase, which converts chlorophyll to chlorophyllide, was found not to be essential for dephytylation in Arabidopsis thaliana. Here, we identify pheophytinase (PPH), a chloroplast-located and senescence-induced hydrolase widely distributed in algae and land plants. In vitro, Arabidopsis PPH specifically dephytylates the Mg-free chlorophyll pigment, pheophytin (phein), yielding pheophorbide. An Arabidopsis mutant deficient in PPH (pph-1) is unable to degrade chlorophyll during senescence and therefore exhibits a stay-green phenotype. Furthermore, pph-1 accumulates phein during senescence. Therefore, PPH is an important component of the chlorophyll breakdown machinery of senescent leaves, and we propose that the sequence of early chlorophyll catabolic reactions be revised. Removal of Mg most likely precedes dephytylation, resulting in the following order of early breakdown intermediates: chlorophyll --> pheophytin --> pheophorbide. Chlorophyllide, the last precursor of chlorophyll biosynthesis, is most likely not an intermediate of breakdown. Thus, chlorophyll anabolic and catabolic reactions are metabolically separated.

During leaf senescence and fruit ripening, chlorophyll is degraded in a multistep pathway into linear tetrapyrroles called phyllobilins. A key feature of chlorophyll breakdown is the removal of the hydrophobic phytol chain that renders phyllobilins water soluble, an important prerequisite for their ultimate storage in the vacuole of senescent cells. Chlorophyllases had been considered for more than a century to catalyze dephytylation in vivo; however, this was recently refuted. Instead, pheophytinase was discovered as a genuine in vivo phytol hydrolase. While chlorophyllase acts rather unspecifically towards different porphyrin substrates, pheophytinase was shown to specifically dephytylate pheophytin, namely Mg-free chlorophyll. The aim of this work was to elucidate in detail the biochemical and structural properties of pheophytinase. By testing different porphyrin substrates with recombinant pheophytinase from Arabidopsis thaliana we show that pheophytinase has high specificity for the acid moiety of the ester bond, namely the porphyrin ring, while the nature of the alcohol, namely the phytol chain in pheophytin, is irrelevant. In silico modelling of the 3-dimensional structure of pheophytinase and subsequent analysis of site-directed pheophytinase mutant forms allowed the identification of the serine, histidine, and aspartic acid residues that compose the catalytic triad, a classical feature of serine-type hydrolases to which both pheophytinase and chlorophyllase belong. Based on substantial structural differences in the models of Arabidopsis pheophytinase and chlorophyllase 1, we discuss potential differences in the catalytic properties of these two phytol hydrolases.

Recent identification of NYE1/SGR1 brought up a new era for the exploration of the regulatory mechanism of Chlorophyll (Chl) degradation. Cluster analysis of senescence associated genes with putative chloroplast targeting sequences revealed several genes sharing a similar expression pattern with NYE1. Further characterization of available T-DNA insertion lines led to the discovery of a novel stay-green gene CRN1 (Co-regulated with NYE1). Chl breakdown was significantly restrained in crn1-1 under diversified senescence scenarios, which is comparable with that in acd1-20, but much more severe than that in nye1-1. Notably, various Chl binding proteins, especially trimeric LHCP II, were markedly retained in crn1-1 four days after dark-treatment, possibly due to a lesion in disassociation of protein-pigment complex. Nevertheless, the photochemical efficiency of PSII in crn1-1 declined, even more rapidly, two days after dark-treatment, compared to those in Col-0 and nye1-1. Our results suggest that CRN1 plays a crucial role in Chl degradation, and that loss of its function produces various side-effects, including those on the breakdown of Ch-protein complex and the maintenance of the residual photosynthetic capability during leaf senescence.

During leaf senescence, chlorophyll is removed from thylakoid membranes and converted in a multistep pathway to colorless breakdown products that are stored in vacuoles. Dephytylation, an early step of this pathway, increases water solubility of the breakdown products. It is widely accepted that chlorophyll is converted into pheophorbide via chlorophyllide. However, chlorophyllase, which converts chlorophyll to chlorophyllide, was found not to be essential for dephytylation in Arabidopsis thaliana. Here, we identify pheophytinase (PPH), a chloroplast-located and senescence-induced hydrolase widely distributed in algae and land plants. In vitro, Arabidopsis PPH specifically dephytylates the Mg-free chlorophyll pigment, pheophytin (phein), yielding pheophorbide. An Arabidopsis mutant deficient in PPH (pph-1) is unable to degrade chlorophyll during senescence and therefore exhibits a stay-green phenotype. Furthermore, pph-1 accumulates phein during senescence. Therefore, PPH is an important component of the chlorophyll breakdown machinery of senescent leaves, and we propose that the sequence of early chlorophyll catabolic reactions be revised. Removal of Mg most likely precedes dephytylation, resulting in the following order of early breakdown intermediates: chlorophyll --> pheophytin --> pheophorbide. Chlorophyllide, the last precursor of chlorophyll biosynthesis, is most likely not an intermediate of breakdown. Thus, chlorophyll anabolic and catabolic reactions are metabolically separated.