Characterization of Senescence Parameters in pph-1.
(A) Maximum quantum yield of PSII (F_v/F_m) during senescence in Col-0 (gray) and pph-1 (black). For senescence induction, attached leaves were covered with aluminum foil. Data are mean values of a representative experiment with three to six replicates. Error bars indicate se.
(B) Analysis of gene expression during dark-induced senescence in Col-0 and pph-1. ACT2 was used as control. Expression was analyzed with nonsaturating numbers of PCR cycles as shown at the right. The results from one of two independent experiments with similar results are shown. PCR products were separated on agarose gels and visualized with ethidium bromide.
(C) Immunoblot analysis of degradation of photosynthesis-related proteins during dark-induced senescence in Col-0 and pph-1. PAO was used as a marker for senescence induction. Gel loadings are based on equal size of leaf area. LSU, Rubisco large subunit.

Analysis of Photosystem Organization During Senescence in pph-1.
(A) Transmission electron micrographs of plastids from short-day-grown Col-0 and pph-1 before (0 d) and after 6 d of dark-induced senescence. Bars = 1 μm.
(B) Sucrose gradient fractionation of thylakoid membranes from Col-0 and pph-1 before (0 d) and after 5 d of dark-induced senescence. Dodecylmaltoside-solubilized thylakoid membranes corresponding to equal amounts of fresh weight were loaded on each gradient. Gradient fractions (1 to 14) used for further analysis are shown at the right. The position of photosystem subcomplexes, as identified by immunoblot analysis of individual photosystem subunits (see Supplemental Figure 3 online), is indicated at the left. Note that during senescence, the LHCII band separated into two distinct bands in both Col-0 and pph-1.

Analysis of Subcellular PPH Localization.
(A) Transient expression in Arabidopsis mesophyll protoplasts of GFP fused in frame to the C terminus of PPH (PPH-GFP). TIC110-GFP was used as control for chloroplast envelope localization. GFP fluorescence (column 1) and chlorophyll autofluorescence (column 2) were examined by confocal laser scanning microscopy. Column 3, merge of GFP and autofluorescence; column 4, bright field image. Bars = 10 μm.
(B) Import of [³⁵S]-Met–labeled precursor of PPH (pPPH) in Arabidopsis chloroplasts. Import was allowed to proceed for 5, 10, and 15 min and mature PPH (mPPH) accumulated. After 15 min of import, chloroplasts were treated with thermolysin (TL) to digest surface-bound and nonimported precursor. To determine the subchloroplast PPH localization, chloroplasts were lysed and separated into stromal (SN) and membrane (P) fractions. The membrane fraction was subjected to alkaline extraction (Na₂CO₃) and separated into Na₂CO₃-resistant proteins (P) or proteins extractable by this treatment (SN). M, molecular size markers (kD) are indicated at the left. IVT, in vitro translation product; LSU, large subunit of Rubisco; CAB, chlorophyll a/b binding protein.

Tentative Models for Chlorophyll Metabolism and for Chlorophyll-Apoprotein Degradation.
(A) Schematic drawing of chlorophyll biosynthesis and degradation, integrating the findings of this work. In this model, anabolic and catabolic steps do not have overlapping activities but are interconnected through the chlorophyll cycle. Thickness of arrows within the cycle reflects relative activities of respective enzymes as suggested by Rüdiger (2002). CLHs might marginally contribute to chlorophyll degradation (thin red arrows), but the bulk of chlorophyll seems to be catabolized via phein a and PPH (thick red arrows).
(B) Tentative model for the degradation of LHC-chlorophyll complexes during senescence in Col-0 and pph-1. Upon senescence induction in the wild type, SGR, NYC1, and PPH are activated. Acting independently or in concert, these induced proteins cause structural changes and/or partial destabilization of the LHC-chlorophyll complexes, at least in part through chlorophyll b to chlorophyll a conversion catalyzed by NYC1. By contrast, the function of SGR in this process is not resolved yet. These changes allow proteases to access and degrade the apoproteins. In the absence of PPH in pph mutants, LHC complexes are stabilized and not accessible for proteases, possibly because of the retention of phytylated pigments within the complexes. Alternatively, LHCs could be stabilized by preventing/slowing down further chlorophyll b to a conversion as a consequence of phein a accumulation.
CAO, chlide a oxygenase; Chl, chlorophyll. CS, chl synthase; MCS, metal-chelating substance; RCCR, red chlorophyll catabolite reductase.

Identification and Analysis of Candidates for Phytol Hydrolysis in Arabidopsis.
(A) Screening of the TAIR protein database (version 8) for proteins with properties expected for a phytol-hydrolyzing enzyme.
(B) Analysis of senescence-related expression of candidate genes using the Genevestigator Meta-Analyzer tool (Zimmermann et al., 2004). The ratio of mean fluorescence values from senescent leaves (organ #44, number of chips: 3) and adult leaves (organ #42, number of chips: 274) is shown. Values for SGR1 and PAO are shown as reference. A cutoff of 3 was chosen for significantly upregulated genes. Note that one of the 24 candidate genes (At5g47860) was not present on the 22K microarray chip.
(C) Degradation of chlorophyll during dark-induced senescence in mutants of candidate genes. Data are mean values of a representative experiment with three technical replicates. Error bars indicate sd.

Deficiency of PPH Causes a Stay-Green Phenotype.
(A) Gene structure of PPH showing the T-DNA insertion sites of the different pph mutants studied here. For pph-1, the site of T-DNA insertion was verified by sequencing. Position of primers used for isolation of homozygous lines is shown.
(B) Analysis of PPH expression during dark-induced senescence. ACT2 was used as control. Expression was analyzed with nonsaturating numbers of PCR cycles as shown at the right. The results from one of three independent experiments with similar results are shown. PCR products were separated on agarose gels and visualized with ethidium bromide.
(C) Phenotype of three pph mutants after 5 d of dark-induced senescence.
(D) Chlorophyll degradation of pph mutants and pph-1 complemented with a 35S-PPH cDNA construct (pph-1/35S:PPH) during dark-induced senescence. Data are mean values of a representative experiment with three replicates. Error bars indicate sd.
(E) Complementation of the stay-green phenotype of pph-1 with a 35S-PPH cDNA construct. A construct harboring a mutation of the proposed active-site Ser residue (35S-PPH_S221A) did not complement pph-1. Leaves after 5 d of dark-induced senescence are shown.

Analysis of Pigment Composition during Dark-Induced Senescence in pph-1.
(A) HPLC traces (A₆₆₅) of leaf extracts of Col-0 (left) and pph-1 (right) before (0 d) and after 5 d of senescence. HPLC injections correspond to equal amounts of fresh weight. Chl, chlorophyll.
(B) Degradation of chlorophyll a (squares) and chlorophyll b (circles) during senescence of Col-0 (closed) and pph-1 (open).
(C) Ratio of chlorophyll a to chlorophyll b during senescence of Col-0 and different pph mutants.
(D) Amounts of phein a (pheophytin a) accumulating during senescence in Col-0 (gray), pph-1 (black), and pph-1 pao1 (white).
(E) Amount of total NCCs found in Col-0 and pph-1 after 5 d of dark-induced senescence.
Data in (B) to (E) are mean values ± sd of representative experiments each with three replicates.

Analysis of Recombinant and Native PPH.
(A) Coomassie blue–stained SDS-PAGE gel of E. coli cells (BL21 DE3) expressing an N-terminal truncated version of PPH (ΔPPH) fused to MBP (MBP-ΔPPH). In addition, a missense mutation of PPH (MBP-ΔPPH_S221A) was analyzed. A strain expressing free MBP was used as control. After induction with isopropyl-β-D-thiogalactopyranosid for 3 h (I), cells were lysed, and soluble proteins (S) were isolated as described in Methods. U, cells before isopropyl-β-D-thiogalactopyranosid induction. Gel loadings are based on equal amounts of cell cultures at OD₆₀₀ = 1.5. Arrowheads indicate position of recombinant proteins. Molecular size markers (kD) are indicated at the left.
(B) and (C) HPLC analysis of assays employing soluble E. coli lysates expressing recombinant MBP-ΔPPH with chlorophyll (B) or phein (C) as substrate. HPLC traces at A₆₆₅ before (0 min) and after 90 min of incubation at 25°C are shown. Note that only in the case of phein were the respective dephytylated products (pheides) formed. Asterisks indicate the C₁₃ epimers of adjacent peaks. In (B), arrows indicate the expected position of chlide b and chlide a, respectively. Chl, chlorophyll.
(D) Pheide a (left) and pheide b (right) formation from phein in assays with recombinant MBP-ΔPPH before (black) and after (white) 24 h of digestion with factor XA. As controls, the substrate was incubated with the vector control (MBP, gray), recombinant MBP-ΔPPH_S221A (hatched), or without E. coli extract (Phein; dotted). Data are mean ± sd of three independent assays.

Phylogenetic Analysis and Sequence Alignment of PPH Homologs.
(A) Maximum likelihood phylogenetic tree of PPH and related proteins from plants, algae, and cyanobacteria. Branch support values obtained from 1,000 bootstraps are indicated when higher than 50%. Three clades (I, II, and III) are distinguished. For protein accession numbers, see Methods. At, Arabidopsis thaliana; Cr, Chlamydomonas reinhardtii; Nt, Nicotiana tabacum; Ol, Ostreococcus lucimarinus; Os, Oryza sativa; Ot, Ostreococcus taurii; Pp, Physcomitrella patens; Ps, Picea sitchensis; Pt, Phaeodactylum tricornutum; Sco, Synechococcus sp PCC 7002; Scy, Synechocystis sp PCC 6803; Vv, Vitis vinifera; Zm, Zea mays.
(B) Sequence alignment of PPH proteins from different species. Black shading with white letters, gray shading with white letters, and gray shading with black letters reflect 80, 60, and 40% sequence conservation, respectively, with Blosum62 similarity group enabled. A conserved domain (named the PPH domain) containing the proposed active-site Ser residue (arrowhead) is underlined.