Graphic model of the proteins involved in chlorophyll breakdown in higher plants and chemical structures of chlorophyll and chlorophyll catabolites. The protein SGR, involved in stay-green mutations, is suggested to function upstream of the chlorophyll catabolic enzymes by destabilizing the LHC (Park et al., 2007). The enzyme chlorophyll b reductase is involved in maintaining the balance between chlorophylls by catalyzing the conversion of chlorophyll b to a. The catabolic steps of the chlorophyll breakdown pathway, which lead to loss of the typical green color, are outlined together with the structures of chlorophyll and the intermediate breakdown products as follows: (1) Chlase catalyzes the cleavage of the hydrophobic thylakoid-anchoring phytol chain from the chlorophyll porphyrin ring, resulting in the product chlorophyllide; (2) removal of the Mg ion from chlorophyllide, yielding pheophorbide a, has not yet been shown to be an enzymatic step (labeled as Mg-dechelatase); and (3) PaO catalyzes the cleavage of the porphyrin ring, resulting in the RCC, which is further metabolized by additional enzymes downstream, exported to the vacuole, nonenzymatically converted to nonfluorescent chlorophyll catabolites (NCCs), and stored indefinitely.

Expression and processing kinetics of citrus Chlase during ethylene-induced fruit color-break. Mature green lemon fruit were treated with ethylene (20 μL L⁻¹) at 25°C in the dark for 0, 12, 24, 48, 72, or 120 h in a 600-L sealed container. The container was ventilated once every 24 h, followed by injection of fresh ethylene, to maintain CO₂ levels below 1%. The flavedo of the treated and control fruit was peeled, frozen in liquid nitrogen, and ground to a powder. A, Total protein from each time point was acetone precipitated, extracted in USB protein extraction buffer, separated by SDS-PAGE (30 μg protein/lane), blotted, and dressed with anti-citrus Chlase antibodies. Protein bands bound by the Chlase-specific antibodies were visualized by chemiluminescence. Relative molecular weights of the bands detected by the antibody are denoted. B, Representative lemon fruit phenotype was documented for each time point of ethylene treatment.

Citrus Chlase expression versus chlorophyll levels in ethylene-treated lemon fruit monitored by in situ immunofluorescence. Mature green lemon fruit were treated with ethylene (20 μL L⁻¹) at 25°C in the dark for 24 h in a 4-L sealed container. The container was ventilated once after 12 h, followed by injection of fresh ethylene, to maintain CO₂ levels below 1%. The flavedo of the treated fruit was dissected and used for obtaining cross sections (70 μm thick) using a vibratome. Tissue sections were fixed and dressed with affinity-purified anti-citrus Chlase antibody (rabbit) followed by Alexa-488-conjugated (goat anti-rabbit) secondary antibody. Fluorescence was visualized using a laser scanning confocal microscope as described in “Materials and Methods.” Images of representative cells (A and B) are presented as follows: 1, confocal image recorded simultaneously in transmitted and red fluorescence mode (i.e. chlorophyll fluorescence superimposed on the bright-field image); 2, confocal image recorded simultaneously in transmitted and green fluorescence mode (i.e. immunofluorescence resulting from the Alexa-488 secondary antibody, corresponding to detection by anti-Chlase antibody, superimposed on the bright-field image); 3, confocal image recorded simultaneously for transmitted, red, and green fluorescence (i.e. red fluorescence corresponds to chlorophyll autofluorescence, green immunofluorescence resulting from the Alexa-488 secondary antibody, corresponding to detection by anti-Chlase antibody, superimposed on the bright-field image). Scale bar = 10 μm (A and B).

Subcellular localization of Chlase in ethylene-treated citrus fruit peel by in situ immunofluorescence. Mature green lemon fruit were treated with ethylene (20 μL L⁻¹) at 25°C in the dark for 24 h in a 4-L sealed container. The container was ventilated once after 12 h, followed by injection of fresh ethylene, to maintain CO₂ levels below 1%. The flavedo of the treated fruit was dissected and used for obtaining cross sections (70 μm thick) using a vibratome (see “Materials and Methods”). Tissue sections were fixed and dressed with Alexa-488-conjugated goat anti-rabbit secondary antibody as a control (A) or affinity-purified anti-citrus Chlase antibody (rabbit) followed by Alexa-488-conjugated goat anti-rabbit secondary antibody (B and C). Fluorescence was visualized using a laser scanning confocal microscope as described in “Materials and Methods.” Images of representative cells (A and B) are presented as follows: 1, green fluorescence of the Alexa-488 secondary antibody corresponds to detection by anti-Chlase antibody; 2, red fluorescence corresponds to chlorophyll autofluorescence; 3, confocal image recorded simultaneously in transmitted and red fluorescence mode (i.e. chlorophyll fluorescence superimposed on the bright-field image); and 4, confocal image recorded simultaneously for red and green fluorescence (i.e. immunofluorescence resulting from detection by anti-Chlase superimposed on the chlorophyll autofluorescence). Chloroplasts demonstrating green fluorescence (i.e. detection by anti-Chlase antibody) were further analyzed by the confocal microscope colocalization program analysis tool. Images of representative chloroplasts (C) are presented as follows: 1, red fluorescence corresponds to chlorophyll autofluorescence; 2, green fluorescence of the Alexa-488 secondary antibody corresponds to detection by anti-Chlase antibody; and 3, sectors of yellow color indicate colocalization of chlorophyll autofluorescence and immunofluorescence resulting from detection by anti-Chlase antibody. Colocalized sectors in the fluorescing chloroplasts account for 76% of the total fluorescence observed. Scale bar = 10 μm (A and B) and 2 μm (C).

MS-based identification of precursor and mature Chlase versions and the N-terminal processing sites. Mature green lemon fruit were treated with ethylene (20 μL L⁻¹) at 25°C in the dark for 12 or 120 h in a 600-L sealed container. The container was ventilated once every 24 h, followed by injection of fresh ethylene, to maintain CO₂ levels below 1%. The flavedo of the treated and control fruit was peeled, frozen in liquid nitrogen, and ground to a powder. Total protein from each time point was acetone precipitated, extracted in RIPA buffer, and Chlase versions were immunoprecipitated using affinity-purified anti-citrus Chlase antibodies. Putative precursor (approximately 35 kD) and mature (approximately 33 kD) Chlase versions were separated from the antibody by SDS-PAGE, visualized by colloidal Coomassie staining, and excised from the gel. Protein bands were digested with trypsin in-gel and eluted peptides were analyzed by MS/MS. A, The N-terminal tryptic and semi-tryptic peptides detected after trypsinization of the approximately 35-kD and 33-kD protein bands, respectively. Tryptic and semi-tryptic peptides are highlighted on the N-terminal amino acid sequence of Chlase (starting at Met-1). Roman numerals (I, II, III) designate the three different semi-tryptic peptides detected. B, Semi-tryptic peptides I, II, and III observed in the TOF mass spectrum of the HPLC fraction that eluted at 23 min. In each case, the multiple peaks correspond to the normal isotopic distribution (i.e. to the inclusion of 0, 1, 2, or 3 13C atoms in the peptide molecule). Mass of the monoisotopic peaks (zero 13Cs) appears above each peak and above each together with the sequences that were confirmed by MS/MS measurements (Table I). Note the different vertical scales in the three segments; all three peptides were observed in the same mass spectrum, collected at the same time under identical instrumental conditions, so the range of observed intensities of the monoisotopic peaks in the three peptide spectra (from approximately 1,500 counts/bin to almost 40,000 counts/bin) provides an estimation of the quantity of each of the detected peptides.