Partial Purification and Characterization of Red Chlorophyll Catabolite Reductase, a Stroma Protein Involved in Chlorophyll Breakdown

Abstract

Red chlorophyll (Chl) catabolite (RCC) reductase, which catalyzes the reaction of an intermediary Chl catabolite (RCC) in the two-step cleavage reaction of pheophorbide (Pheide) a into primary fluorescent catabolites (pFCCs) during Chl breakdown, was characterized and partially purified. RCC reductase activity was present at all stages of barley leaf development and even in roots. The highest specific activity was found in senescent leaves, which were used to purify RCC reductase 1000-fold. Among the remaining three proteins, RCC reductase activity was most likely associated with a 55-kD protein. RCC reductase exhibited saturation kinetics for RCC, with an apparent Michaelis constant of 0.6 mM. The reaction depended on reduced ferredoxin and was sensitive to oxygen. Assays of purified RCC reductase with chemically synthesized RCC as a substrate yielded three different FCCs, two of which could be identified as the stereoisomeric pFCCs from canola (Brassica napus) (pFCC-1) and sweet pepper (Capsicum annuum) (pFCC-2), respectively. In the coupled reaction with Pheide a oxidase and RCC reductase, either pFCC-1 or pFCC-2 was produced, depending on the plant species employed as a source of RCC reductase. Data from 18 species suggest that the stereospecific action of RCC reductase is uniform within a plant family. ~~~~~~~~~~~~~~Of the enzymes that constitute the pathway of Chl breakdown in senescent leaves (for review, see , PaO deserves special attention for severa1 reasons. It is responsible for the oxygenolytic opening of the porphyrin macrocycle and therefore could be considered to be the center piece of the catabolic system. Indeed, the retention of Chl in senescent leaves of mutant genotypes of Festuca pratensis and Pisum sativum has been demonstrated to be due to deficient PaO activities. Its key role in Chl breakdown is also emphasized by the strictly senescence-specific regulation of its activity . In contrast, other catabolic enzymes appear to be present in the (potentially) active form at a11 stages of leaf development.PaO is located in the envelope of senescent chloroplasts and participates in a reaction in two steps by which Pheide a is converted to a linear tetrapyrrolic product (Fig. 1). As demonstrated in a preceding report (Rodoni et al., 1997), in the first step PaO '

SAUR39, a Small Auxin-Up RNA Gene, Acts as a Negative Regulator of Auxin Synthesis and Transport in Rice

“…The expression of 23 genes that have putative functions in photosynthesis and chlorophyll production was down-regulated in OX plants, with the exception of a putative red chlorophyll catabolite reductase, which has been shown to be involved in chlorophyll breakdown (Rodoni et al, 1997) and was up-regulated. The transcription of five genes with putative roles in senescence was higher in OX plants (Supplemental Table S3), which correlates with an early progression of senescence in the OX plants (Fig.…”

Update on the biochemistry of chlorophyll breakdown

“…thylakoid (NYC1), stroma (NOL, PPH, RCCR) and envelope (PAO) (Scheumann et al 1999;Sato et al 2009;Rodoni et al 1997;Matile and Schellenberg 1996;Schelbert et al 2009). As a consequence, Chl pigment shuttling mechanisms between the thylakoid and the envelope have been postulated in the past (Matile et al 1999;Hörtensteiner 2006).…”

Chlorophyll Breakdown in Senescent Chloroplasts (Cleavage of Pheophorbide a in Two Enzymic Steps)

“…The second FCC is chromatographically identical to Ca-FCC-2, the primary FCC produced from Pheide a by enzyme preparations from the pericarp of ripening fruits of sweet pepper (Moser and In the uncoupled reaction, RCC reductase may be able to interact with its substrate, RCC, without spatial restriction by the oxygenase and, therefore, produce FCCs with different configurations, whereas only one product is formed in the coupled assay. In a second report (Rodoni et al, 1997), we demonstrate that in a coupled assay RCC reductases prepared from different plant sources either produce the Bn-FCC-2 or the Ca-FCC-2 type of primary FCCs. We have not been able to identify the nature of the third and most polar FCC emerging in the uncoupled reaction of RCC reductase.…”