In Vivo Participation of Red Chlorophyll Catabolite Reductase in Chlorophyll Breakdown

Abstract

A central reaction of chlorophyll breakdown, porphyrin ring opening of pheophorbide a to the primary fluorescent chlorophyll catabolite (pFCC), requires pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR), with red chlorophyll catabolite (RCC) as a presumably PAO-bound intermediate. In subsequent steps, pFCC is converted to different fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs). Here, we show that RCCR-deficient Arabidopsis thaliana accumulates RCC and three RCC-like pigments during senescence, as well as FCCs and NCCs. We also show that the stereospecificity of Arabidopsis RCCR is defined by a small protein domain and can be reversed by a single Phe-to-Val exchange. Exploiting this feature, we prove the in vivo participation of RCCR in chlorophyll breakdown. After complementation of RCCR mutants with RCCRs exhibiting alternative specificities, patterns of chlorophyll catabolites followed the specificity of complementing RCCRs. Light-dependent leaf cell death observed in different RCCRdeficient lines strictly correlated with the accumulation of RCCs and the release of singlet oxygen, and PAO induction preceded lesion formation. These findings suggest that RCCR absence causes leaf cell death as a result of the accumulation of photodynamic RCC. We conclude that RCCR (together with PAO) is required for the detoxification of chlorophyll catabolites and discuss the biochemical role(s) for this enzyme.

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

“…PAO activity was monitored in vitro essentially as described (Pružinská et al, 2007) but using 0.06 μg μl -1 of His 6 -AtRCCR (Pružinská et al, 2007), and 100 U of catalase (Sigma) to replace red chl catabolite-forming factor (Pružinská et al, 2005). pFCC formation was followed by reversed-phase HPLC and fluorescence detection (320/450 nm) using 36% (v/v) 50 mM potassium phosphate buffer, pH 7, in methanol as solvent (Pružinská et al, 2005).…”

A Role for TIC55 as a Hydroxylase of Phyllobilins, the Products of Chlorophyll Breakdown during Plant Senescence

“…Indeed, in an identical assay, but using epi-pFCC instead of pheophorbide a as substrate, the polar FCC fraction increased in a timedependent manner ( Figure 2B). To confirm identity of this compound as hydroxy-epi-pFCC, we compared its retention time in HPLC with the retention times of the FCCs accumulating in gerontoplasts of Col-0 plants and of acd-22+At-RCCR-X plants (Pruzinská et al, 2007) that were complemented with the epipFCC-producing "X" version of Arabidopsis RCCR (see Introduction). Coinjection experiments indeed confirmed that the product of in vitro hydroxylation of epi-pFCC is identical to the polar FCC fraction from acd2-2+At-RCCR-X chloroplasts ( Figure 2C).…”

“…PAO, in concert with red chlorophyll catabolite reductase (RCCR), catalyzes the ring-opening reaction of pheophorbide a, the phytol-and Mg-free intermediate of the early part of chlorophyll breakdown, to a primary fluorescent chlorophyll catabolite (pFCC), the primary (fluorescent) phyllobilin that is the precursor of all subsequently formed phyllobilins. The direct product of the PAO reaction, red chlorophyll catabolite, does not accumulate but is immediately reduced at the C15/C16-double bond by RCCR in an intriguing stereospecific manner (Wüthrich et al, 2000;Pruzinská et al, 2007) (for atom and pyrrole numbering of phyllobilins, see the structure of pFCC in Figure 1B). Thus, depending on the source of the enzyme, two C16-stereoisomers, pFCC or epi-pFCC, may occur (Mühlecker et al, , 2000.…”

“…For example, the RCCR of Arabidopsis thaliana (At-RCCR) produces pFCC, while in bell pepper (Capsicum annuum) or tomato (Solanum lycopersicum), epi-pFCC is formed . It was shown in vitro that the stereospecificity of Arabidopsis RCCR is defined by a single amino acid residue, Phe-219, which when mutated to Val changes the specificity (Pruzinská et al, 2007). Likewise, complementation of the accelerated cell death2 (acd2) mutant of Arabidopsis that is deficient in RCCR with an RCCR version "X," where six amino acids of the wild-type sequence including Phe-219 were replaced by the respective residues of the tomato RCCR (acd2-2+At-RCCR-X), caused the exclusive accumulation during senescence of C16-isomerized phyllobilins (Pruzinská et al, 2007).…”

Chlorophyll Breakdown in a Fern—Discovery of Phyllobilin Isomers with a Rearranged Carbon Skeleton

“…However, whereas many basic aspects of the path(s) of their formation remain obscure, key structural elements present in 1 and 2 are compatible with a role of an RCC8b, 15 and of FCCs16 as their precursors. Genomic analysis of ferns has, indeed, provided evidence for proteins related to the enzymes PaO17 and RCCR18 in these seedless higher plants,9 crucial for formation of hypothetical PBs, such as RCC and “primary” FCC. Hence, the biochemical pathway to FCC‐type intermediates may be available in the chloroplasts of ferns.…”