Rice NON-YELLOW COLORING1 Is Involved in Light-Harvesting Complex II and Grana Degradation during Leaf Senescence

Abstract

Chlorophyll degradation is an aspect of leaf senescence, which is an active process to salvage nutrients from old tissues. non-yellow coloring1 (nyc1) is a rice (Oryza sativa) stay-green mutant in which chlorophyll degradation during senescence is impaired. Pigment analysis revealed that degradation of not only chlorophylls but also light-harvesting complex II (LHCII)-bound carotenoids was repressed in nyc1, in which most LHCII isoforms were selectively retained during senescence. Ultrastructural analysis of nyc1 chloroplasts revealed that large and thick grana were present even in the late stage of senescence, suggesting that degradation of LHCII is required for the proper degeneration of thylakoid membranes. Mapbased cloning of NYC1 revealed that it encodes a chloroplast-localized short-chain dehydrogenase/reductase (SDR) with three transmembrane domains. The predicted structure of the NYC1 protein and the phenotype of the nyc1 mutant suggest the possibility that NYC1 is a chlorophyll b reductase. Although we were unable to detect the chlorophyll b reductase activity of NYC1, NOL (for NYC1-like), a protein closely related to NYC1 in rice, showed chlorophyll b reductase activity in vitro. We suggest that NYC1 and NOL encode chlorophyll b reductases with divergent functions. Our data collectively suggest that the identified SDR protein NYC1 plays essential roles in the regulation of LHCII and thylakoid membrane degradation during senescence. INTRODUCTIONThe final step of leaf development is senescence, which is an active process to salvage nutrients from old leaves. Leaf yellowing, which is caused by unmasking of preexisting carotenoids by chlorophyll degradation, is a good indicator of senescence (Matile, 2000). Most chlorophyll exists in protein complexes in leaves, because free chlorophyll photooxidatively damages cells. Chlorophyll a is a component of several protein complexes, including the photosystem I (PSI) and photosystem II (PSII) reaction center complexes and the cytochrome b 6 f complex. Chlorophyll b exists only in the light-harvesting chlorophyll a/b-protein complex (LHCP). LHCP binds chlorophyll a, chlorophyll b, and carotenoids (neoxanthin, violaxanthin, and lutein) (Liu et al., 2004). Chlorophyll b is thought to be important for the stability of LHCP (Bellemare et al., 1982). PSI-associated light-harvesting complex I (LHCI) and PSII-associated LHCII proteins are encoded by the Lhca and Lhcb gene families, respectively. LHCPs are localized in the thylakoid membrane. Lhcb1, -2, and -3 are major LHCII proteins and form trimers, but Lhcb4, -5, and -6 occur as monomers. LHCII is localized predominantly in grana, the stacking region of the thylakoid membrane. LHCII has been thought to play an important role in the formation of grana (Allen and Forsberg, 2001).The chlorophyll synthesis pathway has been well characterized, and most, if not all, genes encoding enzymes involved in chlorophyll synthesis have been isolated (Nagata et al., 2005). On the other hand, the chlorophyll degradation pathway is less...

Stay-green protein, defective in Mendel’s green cotyledon mutant, acts independent and upstream of pheophorbide a oxygenase in the chlorophyll catabolic pathway

“…Hence, the gene mutations of type C mutants were considered to affect the pathway of chl breakdown directly. This has been confirmed and is obvious for pao1 and nyc1, which are affected in catabolic steps of the pathway (Pružinská et al, 2003;Pružinská et al, 2005;Kusaba et al, 2007). The situation is different for sgr/sid mutants, since so far the function of SGR/SID could not unequivocally be demonstrated.…”

Update on the biochemistry of chlorophyll breakdown

“…Genes encoding both enzymes have recently been identified Meguro et al 2011). In Arabidopsis and rice, Chl b reductase is encoded by two genes, NON-YELLOW COLOR-ING1 (NYC1) and NYC1-LIKE (NOL) (Horie et al 2009;Kusaba et al 2007;Sato et al 2009). NYC1 and NOL are members of the family of short-chain dehydrogenases/ reductases and share around 50 % sequence identity.…”

“…1 d C1 stereochemistry refers to the type of pFCC, i.e. pFCC (1) or epi-pFCC (epi), formed in the respective species or genus; nd, not determined e Source of material used for catabolite isolation: E, in vitro enzymatic PAO/RCCR assays; F, fruits; L, leaves f In At-NCC-3, the site of hydroxylation is indicated to be C7 (rather than C8 2 ) g Hv-UCC-1 and Ap-UCC-1 are indicated to be pseudo-enantiomers (Müller et al 2011) b reductase, although Chl b reductase activity could so far not be demonstrated with recombinant NYC1 (Horie et al 2009;Kusaba et al 2007). A possible explanation is the hydrophobic nature of NYC1 proteins; they contain three predicted transmembrane spanning domains, fitting the proposed biochemical features of Chl b reductase.…”

Chlorophyll b degradation by chlorophyll b reductase under high-light conditions

“…Chlorophyll b is reduced to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase (CBR). Two CBR isozymes, NON-YELLOW COLORING 1 (NYC1) and NYC1-Like (NOL), are known in land plants (Kusaba et al 2007). An NYC1-deficient mutant cannot degrade LHCII, suggesting that chlorophyll b degradation triggers LHCII degradation.…”

“…Although the phenotypes of CBR-deficient mutants during senescence have been studied (Kusaba et al 2007;Horie et al 2009;Sato et al 2009), the effect of the light conditions on the degradation activity of chlorophyll b has not yet been determined. In this report, we analyzed mutant plants that are defective in chlorophyll b degradation under a high-light treatment to elucidate the role of chlorophyll b degradation in the regulation of the light-harvesting system.…”