MES16, a member of the methylesterase protein family, specifically demethylates fluorescent chlorophyll catabolites during chlorophyll breakdown in Arabidopsis

Abstract

During leaf senescence, chlorophyll (Chl) is broken down to nonfluorescent chlorophyll catabolites (NCCs). These arise from intermediary fluorescent chlorophyll catabolites (FCCs) by an acid-catalyzed isomerization inside the vacuole. The chemical structures of NCCs from Arabidopsis (Arabidopsis thaliana) indicate the presence of an enzyme activity that demethylates the C13(2)-carboxymethyl group present at the isocyclic ring of Chl. Here, we identified this activity as methylesterase family member 16 (MES16; At4g16690). During senescence, mes16 leaves exhibited a strong ultraviolet-excitable fluorescence, which resulted from large amounts of different FCCs accumulating in the mutants. As confirmed by mass spectrometry, these FCCs had an intact carboxymethyl group, which slowed down their isomerization to respective NCCs. Like a homologous protein cloned from radish (Raphanus sativus) and named pheophorbidase, MES16 catalyzed the demethylation of pheophorbide, an early intermediate of Chl breakdown, in vitro, but MES16 also demethylated an FCC. To determine the in vivo substrate of MES16, we analyzed pheophorbide a oxygenase1 (pao1), which is deficient in pheophorbide catabolism and accumulates pheophorbide in the chloroplast, and a mes16pao1 double mutant. In the pao1 background, we additionally mistargeted MES16 to the chloroplast. Normally, MES16 localizes to the cytosol, as shown by analysis of a MES16-green fluorescent protein fusion. Analysis of the accumulating pigments in these lines revealed that pheophorbide is only accessible for demethylation when MES16 is targeted to the chloroplast. Together, these data demonstrate that MES16 is an integral component of Chl breakdown in Arabidopsis and specifically demethylates Chl catabolites at the level of FCCs in the cytosol.

Figure 1.

Identification of MES16. A, Maximum likelihood phylogenetic tree of radish PPD (RsPPD) and MES proteins from Arabidopsis (At), canola (Bn), barley (Hv), and maize (Zm). Branch support values are based on 100 bootstrap replicates and are indicated when higher than 50%. Three subfamilies as determined by Yang et al. (2008) are circled. For sequence accession numbers, see “Materials and Methods.” B, Analysis of senescence-related expression of the Arabidopsis MES family using the Genevestigator Meta-Analyzer tool (Zimmermann et al., 2004). The ratio of mean fluorescence values from senescent leaves (organ no. 44; number of chips, three) and adult leaves (organ no. 42; number of chips, 274) is shown. The value for PAO is shown as a reference. Asterisks indicate that MES8 and MES20 are not represented on the ATH1 chip used for analysis. C, Analysis of gene expression during dark-induced senescence in Col-0. ACT2 was used as a control. Expression was analyzed with nonsaturating numbers of PCR cycles as shown at the right. PCR products were separated on agarose gels and visualized with ethidium bromide.

Figure 2.

Analysis of recombinant MES16. A and B, HPLC analysis of assays employing E. coli lysate expressing 6xHis-MES16 with Pheide (A) and pFCC (B) as substrate. HPLC traces at A₆₆₅ (A) or A₃₂₀ (B) before (0 min) and after 6 and 12 min of incubation at 25°C are shown. For clarity, only a part of the HPLC traces at A₃₂₀ (B) is shown. The insets show the relative concentrations of formed products (O13⁴-desmethyl Pheide, white circles; pyro-Pheide, white squares; O13⁴-desmethyl pFCC, white triangles). Values are means of three replicates. Error bars indicate sd. C and D, Chemical structures of Pheide a and FCCs, respectively. Relevant carbon atoms and pyrrole rings are indicated in the FCC structure in D. E, Competition assays were performed for 6 min using MeIAA, MeSA, or MeJA at a final concentration of 1.5 mm (100× molar excess to pFCC) and Pheide at a concentration of 150 μm (10×). A relative value of 1 corresponds to 0.05 nmol of O13⁴-desmethyl pFCC being produced during the incubation under the standard conditions as described in “Materials and Methods.” Values are means of three replicates. Error bars indicate sd.

Figure 3.

Deficiency of MES16 does not affect the first steps of Chl degradation. A, Gene structure of MES16 showing the T-DNA insertion sites of two different mes16 mutants studied here. The sites of insertion were verified by sequencing. The positions of primers used for RT-PCR are shown. B, Analysis of gene expression during dark-induced senescence in mes16-1 and -2. ACT2 was used as a control. Expression was analyzed with nonsaturating numbers of PCR cycles as shown at the right. After 30 PCR cycles, which produced clearly visible bands in Col-0 (Fig. 1C), MES16 expression was not obvious in mes16 mutants. PCR products were separated on agarose gels and visualized with ethidium bromide. C, Chl degradation of mes16 mutants during dark-induced senescence. Data are mean values of a representative experiment with three replicates. Error bars indicate sd. The asterisks indicate the significance of differences between the wild type and mes16 mutants at P < 0.03 as determined using a two-tailed t test.

Figure 4.

Colorless catabolites of mes16 mutants. A, Colorless catabolites of dark-incubated (10 d) leaves of Col-0 and mes16 mutants were separated by HPLC as described in “Materials and Methods.” A₃₂₀ is shown. For clarity, only a part of the HPLC traces is shown. For identification and peak numbering of FCCs and NCCs, see Table I. B, Relative contents of NCCs and FCCs. Values are means of three replicates. Error bars indicate sd. C, Photographs of dark-incubated (10 d) Col-0 and mes16 leaves under white light and UV light (366 nm). Bar = 1 cm.

Figure 5.

mes16 mutants retain FCCs in the vacuoles. A, Protoplasts and vacuoles were isolated from mes16-1, and colorless catabolites were separated by HPLC as described in “Materials and Methods.” For clarity, only a part of the HPLC traces is shown. For identification and peak numbering of FCCs and NCCs, see Table I. B, Palisade mesophyll of dark-induced leaves (4 d) of Col-0 and mes16-1 observed with a laser scanning confocal microscope. FCC fluorescence was induced with an excitation wavelength of 355 nm, and the emission signal (blue) was recovered between 430 and 470 nm. Red is Chl autofluorescence. Bars = 50 μm. C, FCC-to-NCC isomerization assays performed with pFCC (black squares) or O13⁴-desmethyl pFCC (black circles). Relative concentrations of FCCs and corresponding NCCs are plotted for pH 5 and 6. For more details, see “Materials and Methods.” Calculated half-lives of FCCs are listed in Table II.

Figure 6.

FCCs are the in vivo substrates of MES16. A, Transient expression of MES16-GFP and free GFP in Arabidopsis mesophyll protoplasts. GFP fluorescence (GFP) and Chl autofluorescence (chlorophyll) were examined by confocal laser scanning microscopy. The merge panels show overlays of GFP and autofluorescence. Bars = 20 μm. B, Anti-GFP immunoblotting of proteins from protoplasts expressing MES16-GFP and free GFP. The arrowheads indicate the predicted sizes of transiently expressed proteins. C, The chimeric construct used to target MES16 to the chloroplast. PPH_TP, Amino acids 1 to 48 from PPH, representing the chloroplasts transit peptide; HA, HA tag. D, Verification of MES16 targeting to the chloroplast. Leaves of Col-0 and Col-0/PPH_TP-MES16-HA were fractionated into protoplasts (Pro) and chloroplasts (Chl) and chloroplast subfractions (Mem, chloroplast membranes; Str, stroma). Gel loadings of protoplast and chloroplast fractions are based on equal amounts of chlorophyll. Anti-HA antibodies were used for detection of the chimeric protein. E, Quantification of Pheide a (white bars) and pyro-Pheide a (black bars) in dark-induced (5 d) Col-0, mes16-1, pao1, mes16-1pao1, and pao1/PPH_TP-MES16-HA. Values are means of three replicates, and error bars represent sd. n.d., Not detected.