Molecular Mechanisms Preventing Senescence in Response to Prolonged Darkness in a Desiccation-Tolerant Plant.

The desiccation-tolerant plant Haberlea rhodopensis can withstand months of darkness without any visible senescence. Here, we investigated the molecular mechanisms of this adaptation to prolonged (30 d) darkness and subsequent return to light. H. rhodopensis plants remained green and viable throughout the dark treatment. Transcriptomic analysis revealed that darkness regulated several transcription factor (TF) genes. Stress- and autophagy-related TFs such as ERF8, HSFA2b, RD26, TGA1, and WRKY33 were up-regulated, while chloroplast- and flowering-related TFs such as ATH1, COL2, COL4, RL1, and PTAC7 were repressed. PHYTOCHROME INTERACTING FACTOR4, a negative regulator of photomorphogenesis and promoter of senescence, also was down-regulated. In response to darkness, most of the photosynthesis- and photorespiratory-related genes were strongly down-regulated, while genes related to autophagy were up-regulated. This occurred concomitant with the induction of SUCROSE NON-FERMENTING1-RELATED PROTEIN KINASES (SnRK1) signaling pathway genes, which regulate responses to stress-induced starvation and autophagy. Most of the genes associated with chlorophyll catabolism, which are induced by darkness in dark-senescing species, were either unregulated (PHEOPHORBIDE A OXYGENASE, PAO; RED CHLOROPHYLL CATABOLITE REDUCTASE, RCCR) or repressed (STAY GREEN-LIKE, PHEOPHYTINASE, and NON-YELLOW COLORING1). Metabolite profiling revealed increases in the levels of many amino acids in darkness, suggesting increased protein degradation. In darkness, levels of the chloroplastic lipids digalactosyldiacylglycerol, monogalactosyldiacylglycerol, phosphatidylglycerol, and sulfoquinovosyldiacylglycerol decreased, while those of storage triacylglycerols increased, suggesting degradation of chloroplast membrane lipids and their conversion to triacylglycerols for use as energy and carbon sources. Collectively, these data show a coordinated response to darkness, including repression of photosynthetic, photorespiratory, flowering, and chlorophyll catabolic genes, induction of autophagy and SnRK1 pathways, and metabolic reconfigurations that enable survival under prolonged darkness.

A Role for TIC55 as a Hydroxylase of Phyllobilins, the Products of Chlorophyll Breakdown during Plant Senescence.

Chlorophyll degradation is the most obvious hallmark of leaf senescence. Phyllobilins, linear tetrapyrroles that are derived from opening of the chlorin macrocycle by the Rieske-type oxygenase PHEOPHORBIDE a OXYGENASE (PAO), are the end products of chlorophyll degradation. Phyllobilins carry defined modifications at several peripheral positions within the tetrapyrrole backbone. While most of these modifications are species-specific, hydroxylation at the C32 position is commonly found in all species analyzed to date. We demonstrate that this hydroxylation occurs in senescent chloroplasts of Arabidopsis thaliana. Using bell pepper (Capsicum annuum) chromoplasts, we establish that phyllobilin hydroxylation is catalyzed by a membrane-bound, molecular oxygen-dependent, and ferredoxin-dependent activity. As these features resemble the requirements of PAO, we considered membrane-bound Rieske-type oxygenases as potential candidates. Analysis of mutants of the two Arabidopsis Rieske-type oxygenases (besides PAO) uncovered that phyllobilin hydroxylation depends on TRANSLOCON AT THE INNER CHLOROPLAST ENVELOPE55 (TIC55). Our work demonstrates a catalytic activity for TIC55, which in the past has been considered as a redox sensor of protein import into plastids. Given the wide evolutionary distribution of both PAO and TIC55, we consider that chlorophyll degradation likely coevolved with land plants.

A liquid chromatography-mass spectrometry platform for the analysis of phyllobilins, the major degradation products of chlorophyll in Arabidopsis thaliana.

During senescence, chlorophyll is broken down to a set of structurally similar, but distinct linear tetrapyrrolic compounds termed phyllobilins. Structure identification of phyllobilins from over a dozen plant species revealed that modifications at different peripheral positions may cause complex phyllobilin composition in a given species. For example, in Arabidopsis thaliana wild-type, eight different phyllobilins have structurally been characterized to date. Accurate phyllobilin identification and quantification, which classically have been performed by high performance liquid chromatography (HPLC) and UV/vis detection, are, however, hampered because of their similar physiochemical properties and vastly differing abundances in plant extracts. Here we established a rapid method for phyllobilin identification and quantification that couples ultra-HPLC with high-resolution/high-precision tandem mass spectrometry. Using Arabidopsis wild-type and mutant lines that are deficient in specific phyllobilin-modifying reactions, we identified a total of 16 phyllobilins, among them two that have not been described before in Arabidopsis. The single and collision-induced dissociation tandem mass spectrometry data of all 16 Arabidopsis phyllobilins were collected in a mass spectrometry library, which is available to the scientific community. The library allows rapid detection and quantification of phyllobilins within and across Arabidopsis genotypes and we demonstrate its potential use for high-throughput approaches and genome-wide association studies in chlorophyll breakdown. By extending the library with phyllobilin data from other plant species in the future, we aim providing a tool for chlorophyll metabolite analysis as a measure of senescence for practical applications, such as post-harvest quality control.

Side-chain modifications of phyllobilins may not be essential for chlorophyll degradation in Arabidopsis.

Disposing efficiently and safely chlorophyll derivatives during senescence requires a coordinated pathway that is well conserved throughout green plants. The PAO/phyllobilin pathway catalyzes the degradation of the chlorophyll during senescence and allows detoxification of the pigment and its subsequent export from the chloroplast. Although most of the chloroplastic reactions involved in chlorophyll degradation are well understood, the diversity of enzymes responsible for downstream modifications of non-phototoxic phyllobilins remains to be explored. More than 40 phyllobilins have been described to date, but only three enzymes catalyzing side-chain reactions have been identified in Arabidopsis thaliana, namely, TIC55, CYP89A9, and MES16. Here, by generating a triple mutant, we evaluate the extent to which these enzymes are influencing the rate and amplitude of chlorophyll degradation at the metabolite as well as its regulation at the transcriptome level. Our data show that major side-chain modifications of phyllobilins do not influence significantly chlorophyll degradation or leaf senescence, letting the physiological relevance of their striking diversity an open question.

Isolation and Detection of the Chlorophyll Catabolite Hydroxylating Activity from Capsicum annuum Chromoplasts.

Hydroxylation of chlorophyll catabolites at the so-called C32 position ( Hauenstein et al., 2016 ) is commonly found in all plant species analyzed to date. Here we describe an in vitro hydroxylation assay using Capsicum annuum chromoplast membranes as a source of the hydroxylating activity, which converts the substrate epi-pFCC (epi-primary Fluorescent Chlorophyll Catabolite) ( Mühlecker et al., 2000 ) to epi-pFCC-OH.