Chlorophyll (Chl) molecules harvest light energy and drive electron transfer and thus play central roles in photosynthesis (Nelson and Yocum, 2006). Chl is actively synthesized during greening when the photosynthetic apparatuses are formed (Tanaka and Tsuji, 1985). In the greening phase, newly synthesized Chl binds to proteins to form various Chl–protein complexes (Shimada et al., 1990). Chl–protein complexes in green plants may be classified into two groups (Barber et al., 2000). The first group comprises Chl a–protein complexes, which form the core antennae of photosystems such as the P700–Chl a–protein complex (CP1) and CP43/CP47 of photosystem (PS) II. The second group comprises light-harvesting Chl a/b–protein complexes (LHCs), which form peripheral antennae of PSI and II (Jansson et al., 1997). These complexes and subunit proteins assemble stoichiometrically to form photosystems, although the detailed mechanisms have not yet been elucidated (Ozawa et al., 2009). During this process, Chl synthesis is finely regulated to supply the Chl required for the formation of photosystems. Chl–protein complexes are turned over to maintain photosynthetic activity (Yu and Vermaas, 1990). Especially in green plants, synthesis and degradation of LHCs plays a crucial role during acclimation to variable light intensities (Tanaka and Melis, 1997). The amount of the LHC of PSII (LHCII) varies in response to light intensity to adjust the antenna size of PSII (Melis, 1999). During senescence, all of the Chl–protein complexes are degraded because they are major nitrogen resources in chloroplasts (Makino et al., 2003). When the Chl–protein complexes are degraded, Chl molecules must enter the degradation pathway because free Chl is a deleterious molecule that generates reactive oxygen species (op den Camp et al., 2003) and so must be converted to non-toxic molecules. Chl degradation has been extensively studied, and, with the exception of a putative Mg dechelatase (MDC), the major enzymes responsible for its degradation have been identified (Hortensteiner, 2006; Hortensteiner and Krautler, 2011). According to a recent report, the first step in degradation of Chl a is extraction of Mg ions from Chl by MDC. The resulting pheophytin a (Phein a) is then dephytylated by pheophytinase (PPH) to pheophorbide a (Pheide a) (Schelbert et al., 2009). Pheide a is then oxidatively ring opened by Pheide a oxygenase (PAO) (Pruzinska et al., 2003); this reaction is followed by reduction of fluorescent Chl catabolites to non-toxic molecules. In contrast, Chl b cannot be directly degraded; a formyl group of Chl b must be converted to a methyl group before degradation because PAO cleaves Pheide a but not Pheide b (Hortensteiner et al., 1995). The first step of conversion of Chl b to Chl a is catalyzed by Chl b reductase (CBR) (Ito et al., 1996), which converts Chl b to 7-hydroxymethyl Chl a (HMChl a). Two CBRs, Non-Yellow Coloring 1 (NYC1) and NYC1 Like (NOL), have been identified in rice (Kusaba et al., 2007; Sato et al., 2009) and Arabidopsis (Horie et al., 2009). HMChl a is then converted to Chl a by HMChl a reductase (HCAR) (Meguro et al., 2011). However, some reports have suggested multiple pathways of Chl b degradation based on the broad substrate specificity of the enzymes. Scheumann et al. (1996) found a reducing activity with metal-free Pheide b, and suggested that reduction occurs after removal of the central magnesium. We also found that recombinant NOL has broad substrate specificity and that it converts Pheide b to 7-hydroxymethyl Pheide a (HMPheide a) (Horie et al., 2009). The actual degradation pathway of Chl b remains to be determined.
All of the Chl in the thylakoid membranes binds to proteins to form Chl–protein complexes (Markwell et al., 1979). We recently reported that Chl b in LHCII can be converted to HMChl a by NOL without degradation of the protein moiety of LHCs, indicating that NOL interacts with LHCs and participates in the initial step of LHC degradation (Horie et al., 2009). This idea was supported by the finding that levels of LHCII did not decrease during senescence in nol nyc1 double mutants (Horie et al., 2009). If CBR catalyzed only free Chl released after digestion of the protein moiety of LHCII, no LHCII proteins would remain in the nol nyc1 double mutant. However, it is still unknown whether Chl b is properly degraded only when it exists in LHCII.
In this report, we first examined the substrate specificity of HCAR in order to determine the Chl b degradation pathway. In contrast to NOL, HCAR reduced HMChl a but not 7-hydroxymethyl Phein a (HMPhein a) and HMPheide a, indicating that the first step of Chl b degradation is not Mg dechelation but instead conversion to HMChl a by NOL. However, when Chl b exists in core antenna complexes such as CP1, Pheide b and HMPheide a accumulate during dark incubation, resulting in cell death, indicating that Chl b is not properly degraded when it exists in complexes other than LHCs. In vitro experiments also indicated that NOL could not catalyze conversion of all the Chl b in the core antenna complexes. Based on these results, we discuss the importance and the mechanisms of Chl b degradation.