When chlorophyll degradation is blocked at the pheophorbide a oxygenase (PAO) step in Arabidopsis, the accumulating porphyrin intermediate pheophorbide a is accompanied by a massive, coordinated jasmonate (JA) response in the nucleus, but the molecular mechanism connecting the chloroplast metabolite to JA biosynthesis and signaling is unknown. The open problem is to identify the retrograde signal chain: whether pheophorbide a itself is the active or mobile signal, which sensors, receptors, transporters, or enzymatic steps transduce chloroplast pheophorbide a status into activation of JA biosynthesis, and whether pheophorbide a accumulation is necessary and sufficient for JA pathway induction during senescence. The question remains open: later work has established adjacent branches (reactive oxygen species (ROS)-executed cell death via the NADPH oxidase RBOHD, JA-driven activation of chlorophyll catabolic genes, singlet-oxygen-triggered JA biosynthesis) but no pheophorbide a sensor, transporter, or causal demonstration has been reported.

Background

During leaf senescence, the green pigment chlorophyll is dismantled in the multi-step PAO/phyllobilin pathway, named after pheophorbide a oxygenase (PAO), the enzyme that opens the porphyrin ring of pheophorbide a (Pheide a), the last intact cyclic tetrapyrrole intermediate of chlorophyll breakdown. Upstream of PAO, the magnesium-dechelatase NON-YELLOWING 1 (NYE1, also called STAY-GREEN, SGR) removes Mg from chlorophyll a to give pheophytin a, and pheophytinase (PPH) cleaves off the phytol side chain to yield Pheide a. Downstream of PAO, the pathway produces linear tetrapyrroles called phyllobilins, which are stored in the vacuole. Because several cyclic intermediates are phototoxic, their levels are tightly controlled; loss-of-function mutants such as pao1 (which accumulates Pheide a), nye1-1, and pph-1 retain chlorophyll and are known as stay-green mutants. Senescence is routinely studied by incubating detached Arabidopsis leaves in darkness (dark-induced senescence) and monitoring chlorophyll loss, gene expression, and hormone levels. Jasmonic acid (JA) is an oxylipin (oxygenated fatty-acid-derived) hormone built from the fatty acid alpha-linolenic acid in the chloroplast by enzymes including 13-lipoxygenase (LOX2), allene oxide synthase/cyclase, and 12-oxo-phytodienoic acid (OPDA) reductase 3 (OPR3); its bioactive conjugate jasmonoyl-isoleucine (JA-Ile) is perceived by the F-box receptor CORONATINE-INSENSITIVE1 (COI1), which targets the JAZ (jasmonate ZIM-domain) transcriptional repressors for degradation, freeing MYC transcription factors. JA promotes senescence and directly activates chlorophyll catabolic genes through MYC2/3/4 and the NAC transcription factors ANAC019/055/072 (Zhu et al., 2015), so the hormone-to-degradation direction of a proposed feedback loop is already established. In the reverse direction, chloroplast-to-nucleus (retrograde) signaling is known to use tetrapyrrole-related signals on the biosynthetic side: for example, the heme oxygenase 1-deficient mutant hy1/gun2 accumulates protoporphyrin IX and constitutively overproduces JA and JA-responsive genes (Zhai et al., 2007), although also without a mechanism. Aubry et al. (2020) added the catabolic side of this picture: during dark-induced senescence of pao1, Pheide a accumulates to high levels and phyllobilins disappear, JA rises roughly tenfold over the wild type (to about 2 nmol per gram fresh weight) together with JA-Ile, JA-biosynthesis genes (LOX2, AOC1, AOC2, OPR3) and JA-signaling genes are strongly induced, and the JAZ expression pattern is inverted, whereas the upstream mutants nye1-1 and pph-1, which do not detectably accumulate Pheide a, show almost none of these responses. Time-course profiling of fatty-acid peroxidation products (oxylipins) showed no difference between pao1 and the wild type, arguing against bulk membrane destruction as the cause of the JA burst. The authors therefore proposed a working model in which Pheide a, positioned at the pathway bottleneck, acts as a feed-forward retrograde signal of chloroplast metabolic status that elevates JA biosynthesis, which in turn modulates chlorophyll catabolic genes and the pace of degradation. The model, however, is correlational: no intervention (Pheide a application, epistasis with JA-pathway mutants, or identification of a sensor) was performed, and the paper explicitly states that the exact molecular mechanism, in particular the nature of the retrograde signal(s) linking chloroplast Pheide a sensing to nuclear gene-expression changes, remains to be identified.

Problem Statement

Identify the molecular mechanism - the retrograde signal chain - by which chloroplast pheophorbide a (Pheide a) status is sensed and transduced into activation of jasmonate biosynthesis and signaling during dark-induced senescence in Arabidopsis thaliana. A complete answer must resolve, as jointly supporting this single objective: (i) whether pheophorbide a itself is the active (and, if the signal must leave the chloroplast, mobile) signaling molecule, or whether its accumulation causes the production of other mobile signaling molecules that activate the JA pathway; (ii) the molecular components that connect chloroplast pheophorbide a status to JA biosynthesis activation - the receptor(s)/sensor(s), transporter(s), or enzymatic step(s) involved; and (iii) the causal chain, i.e. a proposed mechanism together with the interventions that would establish that pheophorbide a accumulation is necessary and sufficient for JA pathway induction - for example, pheophorbide a manipulation in wild-type and pathway-mutant backgrounds or epistasis analysis with JA-biosynthesis and JA-perception mutants - with JA and JA-Ile (jasmonoyl-isoleucine, the bioactive JA conjugate) quantification and JA-responsive gene expression as the required readouts. Answers that resolve only one branch (for example, only the cell-death execution downstream of pheophorbide a, or only the JA-to-catabolic-gene arm) do not close the problem.

References

  1. Aubry S, Fankhauser N, Ovinnikov S, Pružinská A, Stirnemann M, Zienkiewicz K, Herrfurth C, Feussner I, Hörtensteiner S (2020) Pheophorbide a May Regulate Jasmonate Signaling during Dark-Induced Senescence. Plant Physiology 182(2):776-791. doi:10.1104/pp.19.01115. https://doi.org/10.1104/pp.19.01115
  2. Guo Y, Ren G, Zhang K, Li Z, Miao Y, Guo H (2021) Leaf senescence: progression, regulation, and application. Molecular Horticulture 1:5. doi:10.1186/s43897-021-00006-9. https://doi.org/10.1186/s43897-021-00006-9
  3. Yu Y, Zhang Q, Sun S, Yang J (2022) Upregulated expression of RESPIRATORY BURST OXIDASE HOMOLOG D underlies lesion-mimic phenotype in dark-treated Arabidopsis pheide a oxygenase mutant leaves. Planta 255(6):110. doi:10.1007/s00425-022-03895-2. https://doi.org/10.1007/s00425-022-03895-2
  4. Hu X, Zeng C, Su J, Khan I, Zada A, Jia T (2021) Overexpressing 7-Hydroxymethyl Chlorophyll a Reductase Alleviates Non-Programmed Cell Death during Dark-Induced Senescence in Intact Arabidopsis Plants. Biomolecules 11(8):1143. doi:10.3390/biom11081143. https://doi.org/10.3390/biom11081143
  5. Ono K, Kimura M, Matsuura H, Tanaka A, Ito H (2019) Jasmonate production through chlorophyll a degradation by Stay-Green in Arabidopsis thaliana. Journal of Plant Physiology 238:53-62. doi:10.1016/j.jplph.2019.05.004. https://doi.org/10.1016/j.jplph.2019.05.004
  6. Zhai Q, Li C-B, Zheng W, Wu X, Zhao J, Zhou G, Jiang H, Sun J, Lou Y, Li C (2007) Phytochrome Chromophore Deficiency Leads to Overproduction of Jasmonic Acid and Elevated Expression of Jasmonate-Responsive Genes in Arabidopsis. Plant and Cell Physiology 48(7):1061-1071. doi:10.1093/pcp/pcm076. https://doi.org/10.1093/pcp/pcm076
  7. Zhu X, Chen J, Xie Z, Gao J, Ren G, Gao S, Zhou X, Kuai B (2015) Jasmonic acid promotes degreening via MYC2/3/4- and ANAC019/055/072-mediated regulation of major chlorophyll catabolic genes. The Plant Journal 84(3):597-610. doi:10.1111/tpj.13030. https://doi.org/10.1111/tpj.13030
  8. Hirashima M, Tanaka R, Tanaka A (2009) Light-Independent Cell Death Induced by Accumulation of Pheophorbide a in Arabidopsis thaliana. Plant and Cell Physiology 50(4):719-729. doi:10.1093/pcp/pcp035. https://doi.org/10.1093/pcp/pcp035
  9. Muñoz P, Munné-Bosch S (2020) Oxylipins in plastidial retrograde signaling. Redox Biology 37:101717. doi:10.1016/j.redox.2020.101717. https://doi.org/10.1016/j.redox.2020.101717
  10. Geng R, Li X, Huang J, Zhou W (2024) The chloroplast singlet oxygen-triggered biosynthesis of salicylic acid and jasmonic acid is mediated by EX1 and GUN1 in Arabidopsis. Plant, Cell & Environment 47(8):2852-2864. doi:10.1111/pce.14910. https://doi.org/10.1111/pce.14910
  11. Tanaka A, Ito H (2025) Chlorophyll Degradation and Its Physiological Function. Plant and Cell Physiology 66(2):139-152. doi:10.1093/pcp/pcae093. https://doi.org/10.1093/pcp/pcae093
  12. Mu Z, Zhou H, Shi X, Ren G, Wen T, Liu S, Yang J (2026) Integrated Transcriptome and Metabolome Analysis Reveals Defense Mechanisms and Key Antifungal Metabolites in Camellia oleifera under Sodium Pheophorbide a Treatment. Journal of Agricultural and Food Chemistry 74(26):20760-20776. doi:10.1021/acs.jafc.6c01604. https://doi.org/10.1021/acs.jafc.6c01604
  13. Li Y, Cao T, Guo Y, Grimm B, Li X, Duanmu D, Lin R (2025) Regulatory and retrograde signaling networks in the chlorophyll biosynthetic pathway. Journal of Integrative Plant Biology 67(4):887-911. doi:10.1111/jipb.13837. https://doi.org/10.1111/jipb.13837