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

Abstract

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.

Keywords: Arabidopsis thaliana; CYP89A9; MES16; TIC55; chlorophyll; phyllobilins; senescence.

FIGURE 1

Chlorophyll (left) is degraded through the PAO/phyllobilin pathway and produces a diverse range of linear tetrapyrroles: the phyllobilins (right). In Arabidopsis, only enzymes modifying side chains of phyllobilins have been identified: TIC55 catalyzing C3² hydroxylation, MES16 catalyzing C8² carboxymethyl group demethylation, and CYP89a9 catalyzing C1 deformylation.

FIGURE 2

(a) The triple mutant cmt does not show any visible phenotype compared with WT or single mutants. pao1 shows slightly chlorotic phenotypes due to the accumulation of pheophorbide a accumulation (Aubry et al., ; Pružinská et al., 2005). (b) The triple mutant cmt degrades chlorophyll as quickly as WT when dark incubated. Error bars indicate SD, n = 18 leaves per genotype. There are no statistical differences between WT and the cmt mutant at any time point studied (unpaired t‐test, p 0.05). (c) The triple mutant cmt does not show any significant differences in electrolyte leakage compared with WT. pao1 and acd2‐1, accumulating pheophorbide a and RCCR, respectively, are used as positive controls.

FIGURE 3

(a) Extracted ion chromatogram of wild‐type phyllobilins (top panel) shows the accumulation of diverse phyllobilins. On the contrary, cmt mutant (bottom panel) accumulates unmodified and nonfluorescent chlorophyll catabolites. MS/MS fragmentation pattern and chemical structure of these two phyllobilins (F_628 and N_628) are shown in Figure S1. (b) Rosettes of 6‐week‐old short‐day grown Arabidopsis and detached leaves that were dark incubated for senescence induction. (c) Methanol extracts of the 7‐dDD wild‐type (Col‐0) and triple mutant cyp mes tic leaf.

FIGURE 4

RNA‐seq profiling of cyp89a9 mes16 tic55 single and cmt triple mutants shows few modifications of the transcriptome during dark‐induced senescence compared with wild type. (a,b) Venn diagrams showing common patterns of differential expression (0 vs. 3 dDD) of down‐ and upregulated genes during dark‐induced senescence, respectively. WT, wild type. (c,d) Venn diagrams showing common patterns of differential expression at 3 dDD across genotypes for down‐ and upregulated genes compared with WT, respectively. (e) Major enriched GO terms identified in the three CCG mutants during dark‐induced senescence (0 vs. 3 dDD) using the Wilcoxon test implemented within the program Pageman (Usadel et al., 2006).

FIGURE 5

Expression of marker genes of the chlorophyll degradation pathway at 0 dDD (white bars) and 3 dDD (black bars). Values are expressed in fragments per kilobase of transcript per million fragments mapped (FPKM). Asterisks represent statistically significant differences pval ≤ .05, fdr ≤ .05 [*] according to DESeq2.