Abstract
Nonfluorescent chlorophyll catabolites (NCCs) were described as products of chlorophyll breakdown in Arabidopsis thaliana. NCCs are formyloxobilin-type catabolites derived from chlorophyll by oxygenolytic opening of the chlorin macrocycle. These linear tetrapyrroles are generated from their fluorescent chlorophyll catabolite (FCC) precursors by a nonenzymatic isomerization inside the vacuole of senescing cells. Here, we identified a group of distinct dioxobilin-type chlorophyll catabolites (DCCs) as the major breakdown products in wild-type Arabidopsis, representing more than 90% of the chlorophyll of green leaves. The molecular constitution of the most abundant nonfluorescent DCC (NDCC), At-NDCC-1, was determined. We further identified cytochrome P450 monooxygenase CYP89A9 as being responsible for NDCC accumulation in wild-type Arabidopsis; cyp89a9 mutants that are deficient in CYP89A9 function were devoid of NDCCs but accumulated proportionally higher amounts of NCCs. CYP89A9 localized outside the chloroplasts, implying that FCCs occurring in the cytosol might be its natural substrate. Using recombinant CYP89A9, we confirm FCC specificity and show that fluorescent DCCs are the products of the CYP89A9 reaction. Fluorescent DCCs, formed by this enzyme, isomerize to the respective NDCCs in weakly acidic medium, as found in vacuoles. We conclude that CYP89A9 is involved in the formation of dioxobilin-type catabolites of chlorophyll in Arabidopsis.
Highlights
Senescence, the ultimate phase of leaf development in higher plants, is phenotypically defined by leaf yellowing, which results from the quantitative loss of chlorophyll
We show that, in Arabidopsis, nonfluorescent chlorophyll catabolites (NCCs) are only minor products of chlorophyll breakdown and up to 90% of chlorophyll is metabolized to a different type of catabolites, termed here as dioxobilin-type chlorophyll catabolite (DCCs)
nonfluorescent DCC (NDCC) were observed as presumed chlorophyll degradation products (Losey and Engel, 2001; Djapic and Pavlovic, 2008; Djapic et al, 2009; Müller et al, 2011), but their means of formation remained unclear
Summary
Senescence, the ultimate phase of leaf development in higher plants, is phenotypically defined by leaf yellowing, which results from the quantitative loss of chlorophyll. Chlorophyll has been shown to be broken down in a multistep pathway to a group of structurally similar colorless linear tetrapyrroles, termed nonfluorescent chlorophyll catabolites (NCCs) (Kräutler and Matile, 1999; Moser et al, 2009; Hörtensteiner and Kräutler, 2011). Thereby, the C5-carbon atom bridging pyrrole rings A and B is retained as formyl group attached to ring B (Kräutler et al, 1991) (Figure 1) This is in contrast with heme degradation by heme oxygenase, where the corresponding carbon atom is lost as CO, The pathway of chlorophyll breakdown can be divided into two parts: early reactions that take place within senescing chloroplasts and that result in the formation of a colorless primary fluorescent chlorophyll catabolite (pFCC) (Mühlecker et al, 1997). Depending on the plant species as the source of red chlorophyll catabolite reductase one of two stereoisomers (i.e., pFCC and epi-pFCC) is formed (Mühlecker et al, 1997, 2000; Pruzinská et al, 2007)
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