Proline oxidation fuels mitochondrial respiration during dark-induced leaf senescence in Arabidopsis thaliana

Abstract

Leaf senescence is a form of developmentally programmed cell death that allows the remobilization of nutrients and cellular materials from leaves to sink tissues and organs. Among the catabolic reactions that occur upon senescence, little is known about the role of proline catabolism. In this study, the involvement in dark-induced senescence of proline dehydrogenases (ProDHs), which catalyse the first and rate-limiting step of proline oxidation in mitochondria, was investigated using prodh single- and double-mutants with the help of biochemical, proteomic, and metabolomic approaches. The presence of ProDH2 in mitochondria was confirmed by mass spectrometry and immunogold labelling in dark-induced leaves of Arabidopsis. The prodh1 prodh2 mutant exhibited enhanced levels of most tricarboxylic acid cycle intermediates and free amino acids, demonstrating a role of ProDH in mitochondrial metabolism. We also found evidence of the involvement and the importance of ProDH in respiration, with proline as an alternative substrate, and in remobilization of proline during senescence to generate glutamate and energy that can then be exported to sink tissues and organs.

Keywords: Arabidopsis thaliana; dark-induced leaf senescence; mitochondria; primary metabolism; proline dehydrogenase; proline metabolism.

Fig. 1.

Chlorophyll contents and proline accumulation during dark-induced leaf senescence in Arabidopsis. (A) Total chlorophyll content was measured in a time-course experiment over 6 d of dark-induced senescence in Col-0 (wild-type, WT), prodh1, prodh2, and the prodh1 prodh2 double-mutant. Data are means (±SE) of four independent rosettes from two independent experiments. The time periods T₀, T₂, and T₃ are delineated by the progression of senescence in leaves according to Chrobok et al. (2016). Variations within each time-point were not statistically different (ANOVA, P>0.05). Different letters indicate significant differences in overall chlorophyll content between time-points (ANOVA, P<0.05). (B) Proline content was measured in the same time-course experiments as shown in (A). Data are means (±SE) of four independent experiments. Different letters indicate significant differences between means on individual days (ANOVA, P<0.05).

Fig. 2.

ProDH expression during dark-induced leaf senescence in Arabidopsis. (A) ProDH1 and ProDH2 expression in the Col-0 wild-type (WT) was quantified by droplet digital PCR after 0–4 d of dark-induced senescence. Data are means (±SE) of four biological replicates. (B) Western blots of total proteins extracted from 100 mg fresh leaves from Col-0 (WT), prodh1, prodh2, and the prodh1 prodh2 double-mutant in a time-course experiment over 6 d of dark-induced senescence. Proteins were separated by SDS/PAGE and blots were probed with an anti-ProDH1 antibody. (This figure is available in colour at JXB online.)

Fig. 3.

ProDH expression in Arabidopsis leaf mitochondria after 5 d of dark-induced senescence. Antibodies raised against recombinant ProDH1 and ProDH2 isoforms were used for western blots of proteins from crude mitochondria extracted from Col-0 (wild-type, WT), prodh1, prodh2 and the prodh1 prodh2 double-mutant (DM). The blots were loaded with 5 µg (ProDH1) and 10 µg (ProDH2) of crude mitochondrial extracts. (This figure is available in colour at JXB online.)

Fig. 4.

Identification of the ProDH2 isoform in Arabidopsis mitochondria by mass spectrometry using crude mitochondrial extracts from leaves of prodh1 plants after 5 d of dark-induced senescence. (A) Summary of MS results. (B) The ProDH2 protein sequence with the identified peptides marked in red.

Fig. 5.

Immunolocalization using antibodies directed against ProDH during dark-induced senescence in leaves of Arabidopsis. The electron micrographs show sections of leaves after 5 d of dark-induced senescence for (A) Col-0 (wild-type, WT), (B) prodh1, (C) prodh2, and (D) the prodh1 prodh2 double-mutant, and show typical labelling within the mitochondria. Arrows indicate the presence of ProDH. No signal was detected in prodh1 prodh2. m, mitochondrion; cp, chloroplast.

Fig. 6.

Analysis of the proline metabolism enzymes P5CS and P5CDH in Arabidopsis leaves over 5 d of dark-induced senescence. Western blots of total proteins extracts taken from Col-0 (wild-type, WT) and the prodh1 prodh2 double-mutant. The blots were obtained using antibodies raised against P5CS and P5CDH recombinant enzymes. (This figure is available in colour at JXB online.)

Fig. 7.

ProDH activity in Arabidopsis leaves in response to dark-induced senescence. Activity was measured in crude mitochondria extracts from leaves 7, 8, and 9 from the base of the plant that were sampled at 0 d and 5 d from Col-0 (wild-type, WT) and the prodh1 prodh2 double-mutant. Data are means (±SE) of at least for independent biological experiments. Different letters indicate significant differences between means as determined by ANOVA followed by Tukey's test (P<0.05).

Fig. 8.

Oxygen consumption in mitochondria isolated from Arabidopsis leaves from Col-0 (wild-type, WT) and the prodh1 prodh2 double-mutant (DM) in response to dark-induced senescence. Oxygen consumption was determined using a Clark-type electrode in crude mitochondria extracts from leaves 7, 8, and 9 from the base of the plant sampled at 0 d and 5 d of dark-induced senescence. The capacity of the cytochrome pathway (CP) was measured from (A) complex I, (B) complex II, and (D) proline. The capacity of the alternative oxidase pathway (AOX) was measured in WT mitochondria from (C) complex II and (D) proline. Data are means (±SE) of at least four biological replicates. Different letters indicate significant differences between means as determined using ANOVA (P<0.05).

Fig. 9.

Primary metabolism pathways of metabolites measured in detached leaves of Arabidopsis under light or dark conditions compared between Col-0 (wild-type) and the prodh1 prodh2 double-mutant. Heat-maps are shown for changes in metabolites in pooled samples of leaves 7–9 from the base of the plant after 5 d under light (left square) or dark (right square) conditions: the blue-to-yellow scale shows the log₂ ratio of the fold-change between the double-mutant and the wild-type. Data represents the mean values of four biological replicates for each time-point.