Unraveling delta1-pyrroline-5-carboxylate-proline cycle in plants by uncoupled expression of proline oxidation enzymes

Abstract

The two-step oxidation of proline in all eukaryotes is performed at the inner mitochondrial membrane by the consecutive action of proline dehydrogenase (ProDH) that produces Delta(1)-pyrroline-5-carboxylate (P5C) and P5C dehydrogenase (P5CDH) that oxidizes P5C to glutamate. This catabolic route is down-regulated in plants during osmotic stress, allowing free Pro accumulation. We show here that overexpression of MsProDH in tobacco and Arabidopsis or impairment of P5C oxidation in the Arabidopsis p5cdh mutant did not change the cellular Pro to P5C ratio under ambient and osmotic stress conditions, indicating that P5C excess was reduced to Pro in a mitochondrial-cytosolic cycle. This cycle, involving ProDH and P5C reductase, exists in animal cells and now demonstrated in plants. As a part of the cycle, Pro oxidation by the ProDH-FAD complex delivers electrons to the electron transport chain. Hyperactivity of the cycle, e.g. when an excess of exogenous l-Pro is provided, generates mitochondrial reactive oxygen species (ROS) by delivering electrons to O(2), as demonstrated by the mitochondria-specific MitoSox staining of superoxide ions. Lack of P5CDH activity led to higher ROS production under dark and light conditions in the presence of Pro excess, as well as rendered plants hypersensitive to heat stress. Balancing mitochondrial ROS production during increased Pro oxidation is therefore critical for avoiding Pro-related toxic effects. Hence, normal oxidation of P5C to Glu by P5CDH is key to prevent P5C-Pro intensive cycling and avoid ROS production from electron run-off.

FIGURE 1.

Summary of Pro synthesis and Pro catabolism pathways in plants. Pro is synthesized from Glu in the cytosol and chloroplasts. The bifunctional enzyme P5CS catalyzes the ATP-dependent phosphorylation of Glu to γ-glutamyl phosphate and then the NADPH-dependent reduction to GSA. P5C and GSA exist in a nonenzymatic equilibrium. GSA/P5C is also generated from ornithine by ornithine aminotransferase (OAT) in the mitochondria and is suggested to serve as a Pro precursor in the cytosol. Following P5C reduction to Pro by P5CR, Pro is transported to the mitochondria and subjected to two-step oxidation by ProDH-FAD complex and P5CDH, both of which are linked to the inner mitochondrial membrane. ARG, arginase.

FIGURE 2.

Transgenic tobacco plants overexpressing MsProDH show an increased Pro oxidation activity. A, RNA blot showing the constitutive high MsProDH expression in leaves of transgenic plants grown under normal conditions (Control, C) or irrigated with 150 mm NaCl for 48 h (S). MsProDH cDNA was used as a probe. B, ProDH-dependent tolerance to the Pro toxic analogue T4C. One-week-old tobacco seedlings were transferred to MS, MS+ 50 mm NaCl, MS + 3 mm T4C, or MS+50 mm NaCl + 3 mm T4C medium and photographed 3 weeks later. Upper panel, WT plants highly sensitive to T4C + NaCl combination (right photo). Lower panel, ProDH-OE plants grown on MS + NaCl + T4C with no toxic effects. C, Pro oxidation assay in WT and ProDH-10 plants. Leaf proteins were extracted from 10-day water-stressed plants. The activity was measured in the presence of increasing Pro concentrations, using NAD⁺ as the electron acceptor. Standard deviation represents three replicates. ProDH-1, -4, -6, and -10 are independent transgenic lines harboring the MsProDH expression cassette.

FIGURE 3.

Comparison of Pro and P5C levels in leaves of salt-stressed WT and MsProDH-overexpressing tobacco plants. Six-week-old plants, grown on soil, were irrigated with 300 mm NaCl. Pro and P5C levels in leaves were measured after 5 weeks of salt irrigation. A, net Pro concentration calculated by subtracting the corresponding P5C amount from the Pro + P5C value obtained by the ninhydrin reaction. B, P5C content determined by O-amino-benzaldehyde assay. The standard deviation represents samples from three different plants of the same line.

FIGURE 4.

Comparison of Pro and P5C content in p5cdh and WT Arabidopsis plants during drought stress and recovery and correlation with the transcript levels of genes involved in Pro biosynthesis and oxidation. Three-week-old WT and p5cdh plants were dehydrated for 72 h and thereafter recovered by adding the water volume lost by evaporation. A, RNA blot analysis of total RNA (10 μg/lane) probed with P5CDH, ProDH, P5CS, and P5CR cDNA fragments. B and C, leaf Pro and P5C content during drought and recovery. The inset in B displays Pro levels normalized to dry weight, showing that during 24 h of water absorption in the recovery period, no loss of Pro is observed in the p5cdh plants. The standard deviation represents three leaf samples, each pooled from 15–20 plants.

FIGURE 5.

Comparison of the levels of Pro and P5C potential precursors in WT and p5cdh plants during drought stress and following recovery. Gas chromatography-mass spectrometry analysis was performed on leaves of WT and p5cdh plants after 72 h of drought or 24 h of recovery as detailed in Fig. 4. The values are calculated relatively to the ribitol internal standard and normalized to the average dry weight value at each time point, as presented in supplemental Table S1. C (control), no stress. The standard deviation represents five leaf samples, each pooled from 15 to 20 plants.

FIGURE 6.

Deficiency in P5C oxidation to Glu increases thermosensitivity. A, the effect of a continuous heat stress on root elongation of young Arabidopsis seedlings of WT and p5cdh plants. The percentage of root elongation during heat stress (38 °C) is expressed relative to nonstressed seedlings (control). The standard deviation represents an average of three repeats of 10 seedlings each. B, basal thermotolerance of WT and p5cdh seedlings to heat stress. Five-day-old seedlings were exposed to 41 °C for 2 h, and survival was estimated 2 days later. The standard deviation represents three repeats, each comprising 100 seedlings.

FIGURE 7.

Exogenous Pro application induces mitochondrial ROS generation in WT and p5cdh roots under dark conditions. Confocal root tip images of 5-day-old WT (A, C, E, and G) and p5cdh (B, D, F, H, and I) seedlings exposed to the following treatments for 24 h in the dark: A and B, control, no treatment; C and D, 100 mm Pro; E and F, 100 μm PQ; G–I, 100 mm Pro + 100 μm PQ. Left column, Numarski images. MitoSox fluorescence images are in green pseudo-color (second from the left). MitoTracker Deep-Red images are in magenta pseudo-color (third from the left), and the merged fluorescence is in the right column. Nuclear localization of MitoSox (I) is shown by costaining with 4′,6-diamidino-2-phenylindole (blue). Bar, 25 mm.

FIGURE 8.

The effect of exogenous Pro application on Pro and P5C content in leaves of p5cdh and WT plants. Three-week-old plants, grown on vermiculite, were treated with MS (Control) or MS containing 100 mm Pro. Free Pro (A) and P5C (B) levels of leaf samples were determined 48 h later. The standard deviation of three replicates is presented.

FIGURE 9.

Pro-induced ROS generation in roots of light grown seedlings. Confocal images of roots of 5-day-old WT (A and C) and p5cdh (B and D) light grown seedlings, untreated (A and B) or treated with 100 mm l-Pro (C and D) for 24 h. Root tips (left, first two columns) and elongation zones (third and fourth columns) are shown. Numarski images are in gray tone (first and third columns) and MitoSox fluorescence in green pseudo-color (second and fourth columns). Bar, 25 mm.

FIGURE 10.

The hypothesized P5C-Pro cycle and its hyperactivity during uncoupled P5C oxidation. In response to abiotic stresses, Pro is gradually accumulated by increased synthesis and suppressed oxidation. Once the stress is relieved, ProDH-FAD complex oxidizes Pro to P5C while transferring electrons to the mtETC. P5C further oxidation to Glu and then to α ketoglutarate (αKG) is coupled to NAD(P)H formation. When Pro is supplied in excess and ProDH transcription and activity are induced, P5CDH activity does not keep up with ProDH activity, and Pro oxidation becomes uncoupled. Under such conditions, the rate of P5C generation in the mitochondria exceeds its further oxidation to Glu. P5C excess is transported to the cytosol and reduced by P5CR to Pro that is transported to the mitochondria. This P5C-Pro intensive cycling elevates the flow of electrons through ProDH-FAD to the mtETC and to O₂, leading to concomitant generation of ROS. In the p5cdh mutant, the intensive operation of this cycle enhances ROS generation and their spreading to the nuclei and very likely induces programmed cell death (PCD). ETC, electron transfer chain; UQ, ubiquinone.