Pheophorbide a May Regulate Jasmonate Signaling during Dark-Induced Senescence

Abstract

Chlorophyll degradation is one of the most visible signs of leaf senescence. During senescence, chlorophyll is degraded in the multistep pheophorbide a oxygenase (PAO)/phyllobilin pathway. This pathway is tightly regulated at the transcriptional level, allowing coordinated and efficient remobilization of nitrogen toward sink organs. Using a combination of transcriptome and metabolite analyses during dark-induced senescence of Arabidopsis (Arabidopsis thaliana) mutants deficient in key steps of the PAO/phyllobilin pathway, we show an unanticipated role for one of the pathway intermediates, i.e. pheophorbide a Both jasmonic acid-related gene expression and jasmonic acid precursors specifically accumulated in pao1, a mutant deficient in PAO. We propose that pheophorbide a, the last intact porphyrin intermediate of chlorophyll degradation and a unique pathway "bottleneck," has been recruited as a signaling molecule of chloroplast metabolic status. Our work challenges the assumption that chlorophyll breakdown is merely a result of senescence, and proposes that the flux of pheophorbide a through the pathway acts in a feed-forward loop that remodels the nuclear transcriptome and controls the pace of chlorophyll degradation in senescing leaves.

Figure 1.

Phenotypic characterization of CCGs mutants during dark-induced senescence of detached leaves. A, Wild-type (WT), pao1, nye1-1, and pph-1 detached leaves before (0) and after 2 and 5 dd. Images of leaves were digitally abstracted and used to make a composite image for comparison. Scale bar = 5 mm. B, Chl degradation of CCG mutants during dark-induced senescence. C, Electrolyte leakage of CCG mutants during dark-induced senescence. D, Profile of the accumulation of pheophorbide a and the major phyllobilin (DNCC_618) in CCG mutants during dark-induced senescence. Data in (B) to (D) are mean values of a representative experiment with three (B), at least 10 (C), and five (D) replicates, respectively. Error bars indicate sd.

Figure 2.

RNA-seq profiling of CCG mutants provide new insight into the relationship of the PAO/phyllobilin pathway to global leaf senescence. A, Major enriched GO terms identified in the three CCG mutants during dark-induced senescence (0 versus 2 dd) using the Wilcoxon test implemented within the program Pageman (Usadel et al., 2006). B, Principal component analysis of the RNA-seq data. C, Venn diagrams showing common patterns of differential expression (0 versus 2 dd) of up- and downregulated genes during dark-induced senescence. WT, wild type.

Figure 3.

Influence of dark-induced senescence on the expression of the genes involved in the PAO/phyllobilin pathway. Heat maps represent log₂ (FC) of gene expression in each of the four studied lines during dark-induced senescence, as indicated in the inset box. Genes/enzymes: CAO, chlorophyll a oxygenase; CHLG, chlorophyll synthase; HCAR, 7-hydroxymethyl chlorophyll a reductase; NOL, NYC1-like; NYC1, nonyellow coloring1 (chlorophyll b reductase); RCCR, RCC reductase; TIC55, translocon at the inner chloroplast envelope55. Phyllobilins: DNCC, dioxobilin-type NCC; NCC, nonfluorescent chlorophyll catabolite; pFCC, primary fluorescent chlorophyll catabolite. WT, wild type.

Figure 4.

Transcriptional regulation of the PAO/phyllobilin pathway during dark-induced senescence is mainly affected in pao1. Heat maps represent log₂ (FC) of gene expression in each of the four studied lines during dark-induced senescence, as indicated in the inset box. Genes/enzymes: ABF, ABA-responsive element binding factor; ABI5, ABA insensitive5; COI1, coronatine insensitive1; EEL, enhance em level; EIN, ethylene insensitive; ELF3, early flowering3; ERF17, ethylene response factor17; MYC, myelocytomastosis; NAC, NAM, ATAF1/2, and CUC2 domain protein; NAP, NAC-like, activated by PA3/PI; ORE1, oresara1; phyB, phytochrome B; PIF, phytochrome interacting factor; PYL9, pyrabactin resistance1-like9; SnRK2, Ser/Thr kinase2; SOC1, suppressor of overexpression of coi1. WT, wild type.

Figure 5.

JA metabolism during dark-induced senescence in wild type (WT) and pao1. Levels of JA and JA-related metabolites in gray (0 dd) and black (2 dd) for wild type and pao1 are shown as histograms, as indicated in the inset box. Data are mean values of five replicates. Error bars indicate sd. Asterisks indicate significant differences (by one-sided t test with P ≤ 0.05). Expression levels are shown using heat maps of log₂ (FC), as indicated in the inset box. Genes/enzymes (in red): AOC, allene oxide cyclase; AOS, allene oxide synthase; CYP, cytochrome P450 monooxygenase; DAD1, delayed anther dehiscence1; IAR3, IAA-Ala resistant3; JAR1, JA-amino acid synthetase1; JMT, jasmonate methyltransferase; JOX, JA-induced oxygenase; OPR3, OPDA reductase 3; ST2a, sulfotransferase 2a. Metabolites that were quantified (in green): dn-OPDA, dinor-12-oxo-phytodienoic acid; JA-Ile, jasmonyl-Ile; JA-Leu, jasmonyl-Leu; JA-Val, jasmonyl-Val; OPC-4, 3-oxo-2-cis-2-pentenyl-cyclopentane-tetranoic acid; OPC-6, 3-oxo-2-cis-2-pentenyl-cyclopentane-hexanoic acid; OPDA, 12-oxo-phytodienoic acid; 12COOH-JA, 12-carboxy-jasmonic acid; 12COOH-JA-Ile, 12-carboxy-jasmonyl-Ile; 12HSO₄-JA, 12-sulfo-jasmonic acid; 12O-Gly-JA, 12-glycosyl-jasmonic acid; 12OH-JA-Ile, 12-hydroxy-jasmonyl-Ile. Further metabolites (in black): JA-CoA, jasmonyl-coenzyme A; Me-JA, methyl-jasmonic acid; OPC-8, 3-oxo-2-cis-2-pentenyl-cyclopentane-octanoic acid; 4,5ddh-JA, 4,5-didehydro-jasmonic acid; 7iso-JA, 7-iso-jasmonic acid; 7isoMe-JA, 7-isomethyl-jasmonic acid; 12OH-JA, 12-hydroxy-jasmonic acid.

Figure 6.

WGCNA sheds new light on the regulation of the PAO/phyllobilin pathway. A, Heat map showing a module-sample association matrix. Each row corresponds to a module. The heat map color code from blue to red (see inset box) indicates the correlation coefficient between the module (first column) and either the treatment (second column = darkness) or the genetic background (wild type [WT], nye1-1, pph-1 or pao1). B, Patterns of gene expression (left) and size (i.e. number of genes; right) of gene coexpression modules. On the left, heat maps indicate mean expression (log₂ [FC]) of the 10% most representative genes (highest connectivity) for each WGCNA module during dark-induced senescence. N/A, not applicable. C, The regulatory network of the PAO/phyllobilin pathway as exported from WGCNA and visualized in VisANT 5.51 (Hu et al., 2007). Larger nodes show the input genes (CCGs, transcriptional regulators according to Fig. 4). Smaller nodes were limited to the top three most connected genes for each input gene. The edges represent connections between the genes. Node colors represent the module in which the genes clustered during WGCNA analysis.

Figure 7.

Model illustrating the influence of pheophorbide a homeostasis on JA signaling. The middle (wild type [WT]) shows the PAO/phyllobilin pathway under normal senescence conditions, leading to the complete degradation of chl to vacuole-localized phyllobilins. Left and right show modulation of catabolite homeostasis caused by mutations of either nye1-1 or pph-1 (left) or pao1 (right), and the respective observed downstream modulation of the JA response (dashed arrows). Arrow sizes schematically represent relative flux (metabolite) and response (JA signaling) intensities. Among the few genes differentially expressed in nye1-1 and pph-1, jasmonate-ZIM domain genes were downregulated compared to wild type. On the other hand, in pao1, JA biosynthesis and signaling genes as well as some JA bioactive derivatives were induced. DNCC, dioxobilin-type nonfluorescent chlorophyll catabolite; NCC, nonfluorescent chlorophyll catabolite; pFCC, primary fluorescent chlorophyll catabolite.