What causes chlorophyll to break down?

Question

What causes chlorophyll to break down?

Answer 1

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 post-translationally regulated as well.

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During leaf senescence, chlorophyll (chl) is degraded to colorless linear tetrapyrroles, termed nonfluorescent chl catabolites (NCCs; refs. 1–3). The pathway of chl catabolism (Fig. 1A) includes the occurrence of both colored and colorless intermediates. Thus, in two subsequent reactions catalyzed by chlorophyllase and Mg dechelatase, respectively, phytol and the central Mg atom are removed. Then, the ring structure of pheophorbide (pheide) a (Fig. 1 A) is oxygenolytically opened at the -mesoposition between C4 and C5 by pheide a oxygenase (PaO). The product, red chl catabolite (RCC), does not accumulate in vivo (4) but is rapidly converted to a primary fluorescent chl catabolite (pFCC) by a stereospecific reduction of the C20/C1 double bond. The source of the responsible enzyme, RCC reductase (RCCR), defines which of two possible C1 isomers, pFCC-1 or -2, occurs (Fig. 1 A). RCCR of Arabidopsis has been shown to produce pFCC-1 (5). Further steps of the chl breakdown pathway involve reactions known from plant detoxification mechanisms (6). FCCs are hydroxylated and in some cases conjugated with a glucosyl or malonyl moiety (7, 8), followed by their export into the vacuole by a primary active ATPase (9). Finally, FCCs are nonenzymically tautomerized to the respective NCCs because of the acidic pH inside the vacuole (10).

The biochemistry of chl catabolism has been investigated extensively during the last years (for recent reviews, see refs. 3, 5, 11, and 12). Surprisingly, PaO turned out to be a key regulator of this pathway. Thus, PaO activity is detectable only during senescence (13, 14), whereas activities of other enzymes, such as chlorophyllase and RCCR, are constitutive (15–17). In addition, the reactions catalyzed by PaO and RCCR are responsible for the loss of pigment color. Biochemical evidence suggests that the two enzymes are interacting during catalysis. Thus in vitro, the intermediate, RCC, does not accumulate to substantial amounts in the absence of RCCR, indicating that RCC is metabolically channeled (4). PaO has been demonstrated to be located at the inner envelope of senescing chloroplasts (18). In contrast, RCCR is a stroma protein, suggesting that pheide a to pFCC conversion occurs at the stromal periphery of the inner envelope (4, 19). The recent cloning of RCCR (20) has uncovered a distinct relationship to other plant bilin reductases, all of which are ferredoxin (Fd)-dependent (21). Reduced Fd is also needed as a source of electrons for the PaO/RCCR-catalyzed reaction (13, 19). PaO is a nonheme iron type (14) monooxygenase that introduces one atom of molecular oxygen at the -methine bridge of pheide a (Fig. 1 A), giving rise to the formyl group attached to pyrrole ring B in pFCC (22). Its activity is restricted to pheide a, with pheide b being a competitive inhibitor. Consequently, all NCCs identified so far are derived from chl a (23). Before entering this degradation pathway, chl b has to be converted to chl a. A respective chlorophyll cycle that is responsible for the interconversion of both types of chl has been described (24, 25). In addition, activity of one of the enzymes involved in chl b to a conversion, chl b reductase, increases during barley leaf senescence (26).

Senescence is the final stage of leaf development, ultimately leading to the death of the entire leaf. It is a highly regulated process that involves an ordered disintegration of chloroplast components, such as thylakoid membranes, along with the remobilization of amino acids from proteins, such as the chl a/b-binding proteins, and the subsequent release of potentially phototoxic chl. It is commonly believed that the function of chl degradation in plants is to avoid this hazard (3). chl in its free form would cause photooxidative damage to the senescing cell, and it has been shown that the accumulation of photoactive intermediates of chl biosynthesis causes a premature cell death phenotype in two chl biosynthetic mutants and transgenic plants (27, 28). This is termed a lesion mimic phenotype that, in contrast to the phenotypically similar hypersensitive response, occurs in the absence of pathogen infection. In addition to chl biosynthetic mutants, Arabidopsis accelerated cell death 2 (acd2) has been isolated that develops a light-dependent lesion mimic phenotype (29). acd2 is deficient in RCCR, and the phenotype has been suggested to be caused by the accumulation of phototoxic RCC (30). Thus, the ability of plants to degrade chl during senescence seems vitally important. Here we describe the molecular identification of PaO. In addition, we show that a mutant that is defective in PaO shows a stay-green phenotype in the dark and accumulates pheide a, which causes light-dependent premature