Chlorophyll degradation begins with disruption of pigment-protein complexes inside of chloroplasts, a process that frees chlorophyll molecules. A protein without enzymatic activity (SGR) interacts with protein thylakoidal complexes, destabilizing their structure, and contributing to the release of chlorophyll; as a prerequisite for the subsequent degradation of both apoprotein and chlorophyll (Hörtensteiner, 2009). Chlorophyll is a photoactive compound that generates free radicals that can damage several cellular components. For this reason chlorophyll must be quickly degraded as a detoxification mechanism (Matile et al., 1999).
The currently accepted biochemical pathway of degradation of chlorophyll comprises two stages, which are divided according to the moment of the tetrapyrrole ring opening (Figure 1).
Products of the first stage (prior to the rupture of the macrocycle) totally or partially retain the green color, while those of the second stage lose that coloration and are almost colorless. The first stage includes modifications to the side chain of the macrocycle, the hydrolysis of phytol, release of Mg2+ from the tetrapyrrole, and other reactions that can vary among species. The second stage is essential to the loss of green color characteristic during senescence. In most of the cases studied, no degradation intermediates were accumulated at a detectable amount, which suggests that a number of catabolic reactions occurred in coordination.
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Figure 1. Pathway of chlorophyll degradation. First Step: before PaO; Second Step: after PaO. Chla:
chlorophyll a; Chlb: chlorophyll b; Chlda: chlorophyllide a; Chldb: chlorophyllide b; Phea: pheophytin a; Pheoa: pheophorbide a; RCC: red chlorophyll catabolite; FCC: fluorescent chlorophyll catabolite;
NCC: non-fluorescent chlorophyll catabolite. SGR: stay green; Chlase: chlorophyllase; MDS:
magnesium dechelatase; PaO: pheophorbide a oxygenase; RCCR: red chlorophyll catabolite reductase.
Once chlorophyll molecules are released, the first catabolic reaction that occurs is the elimination of phytol. Chlorophyllase (chlorophyll-chlorophyllido hydrolase, CLH, EC 3.1.1.14) was the first enzyme to be identified as causing this reaction (Hörtensteiner, 2006;
Lee et al., 2010; Schenk et al., 2007; Tsuchiya T et al., 1999). In many cases, this enzyme is a glycosylated protein associated to hydrophobic chloroplast membranes and other organelles, and is characterized by its functional latency. Although at the outset it was believed that chlorophyllases were located in the chloroplast membrane (Brandis et al., 1996; Matile et al., 1987) no transmembrane domains have been found in the sequences obtained to date, according to the hydropathy profiles. This suggests that chlorophyllases are not intrinsic membrane proteins. The functional property of latency observed in CLH could be simply due to the spatial separation between the enzyme and its substrate. Chlorophyllase catalyzes the hydrolysis of the ester linkage between the chlorophyll and phytol, reaction that is considered as the first step in the catabolism of chlorophyll. The products of such reaction are phytol and chlorophyllide ((Benedetti and Arruda, 2002; Matile et al., 1999; Takamiya et al., 2000).
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Recently, the true involvement of chlorophyllases in chlorophyll breakdown has been questioned, given that not all isolated genes have a chloroplast transit peptide, suggesting alternative pathways occurring outside of the chloroplast or involvement of enzymes, other than CLH (Hörtensteiner, 2006; Takamiya et al., 2000). Schenk et al. (2007) have shown that Arabidopsis mutants with interrupted expression for both known chlorophyllases are still able to degrade chlorophyll during senescence, indicating that these genes are not essential for this catabolic process. Based on these findings, Schelbert et al. (2009) set out to reveal the true CLH responsible for chlorophyll dephytilation in Arabidopsis. Instead, their findings revealed the existence of a new enzyme, termed pheophytinase (PPH), which would act as a pheophytin hydrolase.
Assuming that chlorophyllase is the first enzyme in the catabolic pathway, the next step would be the elimination of the central ion Mg2+ from chlorophyllide. With regard to this step, studies have revealed conflicting data, and for this reason, consensus has not been reached. Two types of activity were found: one associated with a low molecular weight compound stable at high temperatures (Shioi et al., 1996), and other associated with a thermally labile protein that is probably part of chloroplast membranes (Vicentini et al., 1995). The important difference between these two proposals regarding activity can be explained considering that the low molecular mass compound is a cofactor of an enzyme of higher molecular mass (Costa et al., 2002). More recently, the development of new proposals has suggested that protein with Mg-dechelatase activity can only act in vitro on a widely used artificial substrate, chlorophyllin, but not on the in vivo substrate, clorophyllide, while the low molecular weight compound acts on both substrates (Kunieda et al., 2005; Suzuki et al., 2005; Suzuki and Shioi, 2002). However, if chlorophyllases are not the main enzymes that release phytol, and dephytilation occurs on Mg-free chlorophyll, then the whole degradation pathway must be revised, especially with regard the early reactions. In the new suggested model, Mg release seems to precede phytol cleavage, producing pheophytin, which is then dephytilated by PPH to give pheophorbide (Schelbert et al., 2009).
Besides chlorophyllase and Mg-dechelatase, other modifications of chlorophyll before ring opening were described. An enzyme known as pheophorbidase was described and purified from Chenopodium album. This enzyme catalyzes the hydrolysis of methyl ester bond of pheophorbide isocyclic ring. The product of this reaction is not stable; hence it is converted to pyropheophorbide nonenzymatically. An interesting aspect is that pheophorbidase is located outside the chloroplast, indicating that if pheophorbide a is the true substrate of this enzyme, there would be a degradation pathway, the initial steps of which occur outside the chloroplast (Takamiya et al., 2000).
The stage of tetrapyrrole ring opening is crucial for the loss of green color in the tissues and for eliminating the photo-activity of chlorophyll. This reaction is carried out by the enzyme pheophorbide a oxygenase (PAO), which is a component of the inner membrane of gerontoplasts and chromoplasts. PAO catalyzes the oxygenolitic breakdown of the ring between the C4 and C5 by adding two oxygen atoms and four hydrogen atoms. The product of this reaction is the first identifiable colorless compound, RCC (red chlorophyll catabolite).
PAO is a Rieske-type iron-sulfur oxygenase that requires the presence of reduced ferredoxin and NADPH and both compounds are present in the gerontoplasts, but not in presenescent tissues (Hörstensteiner et al., 1998; Pruzinskà et al., 2003; Takamiya et al., 2000). For this reason, it was initially believed that its activity was limited to senescence. However, recent research has suggested that PAO activity is present from before the onset of senescence
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(Pruzinskà et al., 2003; Pruzinská et al., 2005; Roca et al., 2004). The enzyme also has a high specificity towards the pheophorbide a, while the pheophorbide b induces a competitive inhibition (Engel et al., 1996; Iturraspe et al., 1994).
A reduction of the RCC methine bridge occurs coupled with the previously described oxygenase reaction. Such reaction is catalyzed by the enzyme RCC reductase, which is a soluble enzyme localized in the stroma of chloroplasts (Rodoni et al., 1997). This reaction produces a fluorescent colorless compound called pFCC (by primary fluorescent chlorophyll catabolite). Such reaction requires the presence of ferredoxin and the absence of oxygen, indicating that reactions of RCC and PAO are very closely related; due to the fact that the oxygenase reaction consumes oxygen and maintains anaerobic microenvironment for the reductase (Takamiya et al., 2000; Wüthrich et al., 2000).
While the chlorophyll degradation pathway seems to be very similar to the formation of pFCC in different species, due to the wide variety found in these compounds, it may be assumed that a series of reactions occurs after formation. The pFCC are converted to fluorescent chlorophyll catabolites (FCC) with several modifications that vary among species, such as demethylation and hydroxylation (Hörtensteiner, 2006). Most of the enzymes involved in these steps have not yet been identified, although such reactions are known to cause an increase in the solubility of catabolites. Recently, a member of the methylesterase protein family (MES16) has been identified described in Arabidopsis. It specifically demethylates chlorophyll catabolites at the level of FCCs (Christ et al., 2011). It is considered that the modified FCCs are transported to the vacuole by ATP dependent translocators on the tonoplast, and then converted to NCC (nonfluorescent chlorophyll catabolites) through a rearrangement of the double bond tetrapyrrole and its adjacent methine bridge.
Modifications to the NCC are diverse and very much depend on the species. Furthermore, it is not entirely clear whether NCCs are stored in vacuoles without further degradation. In some species accumulated NCC levels correspond to the total degraded chlorophyll, indicating that the NCC is the end of chlorophyll degradation (Hörstensteiner et al., 1998;
Matile et al., 1999). However, in other species, such as tobacco and spinach, NCC concentrations are higher in early stages of leaf senescence, but lower in later stages (Hörtensteiner, 2006).
Chlorophyll b is an accessory pigment in light harvesting complexes that can represent up to 30% of the total chlorophyll. Despite its relative abundance in higher plants, chlorophyll catabolites derive from chlorophyll a. This indicates that there should be a conversion of chlorophyll b to chlorophyll a early in the degradation pathway, which is supported by several observations. For example, enzyme pheophorbide a oxygenase only takes pheophorbide a as substrate, while the pheophorbide b is a competitive inhibitor. The inhibition of pheophorbide a oxygenase causes the accumulation of both forms of chlorophyllide, but only of pheophorbide a. These findings suggest the presence of enzymes with chlorophyll b reductase activities. Recently, it has been described that the conversion of chlorophyll b to chlorophyll a requires two steps. Horie et al. (2009) showed that a gene named AtNYC encodes for a chlorophyll or chlorophyllide b reductase transforming chlorophyll b to 7-hydroxymethyl chlorophyll a. This compound is finally transformed by 7-hydroxymethyl chlorophyll a reductase producing chlorophyll a (Meguro et al., 2011).
All genes encoding the enzymes described above, except Mg-dechelatasa, have been cloned and characterized, particularly in Arabidopsis thaliana (Hörtensteiner and Kräutler, 2011).
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Additionally, an alternative route of chlorophyll catabolism has been also suggested.
Studies conducted on different systems support the possible participation of peroxidase in the catabolism of chlorophyll (Maeda et al., 1998; Martínez et al., 2001; Funamoto et al., 2002;
Funamoto et al., 2003; Costa et al., 2004; Funamoto et al., 2006).