Two alanine aminotranferases link mitochondrial glycolate oxidation to the major photorespiratory pathway in Arabidopsis and rice

Abstract

The major photorespiratory pathway in higher plants is distributed over chloroplasts, mitochondria, and peroxisomes. In this pathway, glycolate oxidation takes place in peroxisomes. It was previously suggested that a mitochondrial glycolate dehydrogenase (GlcDH) that was conserved from green algae lacking leaf-type peroxisomes contributes to photorespiration in Arabidopsis thaliana. Here, the identification of two Arabidopsis mitochondrial alanine:glyoxylate aminotransferases (ALAATs) that link glycolate oxidation to glycine formation are described. By this reaction, the mitochondrial side pathway produces glycine from glyoxylate that can be used in the glycine decarboxylase (GCD) reaction of the major pathway. RNA interference (RNAi) suppression of mitochondrial ALAAT did not result in major changes in metabolite pools under standard conditions or enhanced photorespiratroy flux, respectively. However, RNAi lines showed reduced photorespiratory CO(2) release and a lower CO(2) compensation point. Mitochondria isolated from RNAi lines are incapable of converting glycolate to CO(2), whereas simultaneous overexpression of GlcDH and ALAATs in transiently transformed tobacco leaves enhances glycolate conversion. Furthermore, analyses of rice mitochondria suggest that the side pathway for glycolate oxidation and glycine formation is conserved in monocotyledoneous plants. It is concluded that the photorespiratory pathway from green algae has been functionally conserved in higher plants.

Fig. 1.

Inhibition of mitochondrial glycolate metabolism by aminooxyacetate (AOA). Isolated Arabidopsis mitochondria corresponding to 50 μg of protein were incubated with [¹⁴C]glycolate or [¹⁴C]glycine. AOA (1 mM) was added as inhibitor. ¹⁴CO₂ released was captured in NaOH and determined by scintillation counting. Shown are the means ±SE from three independent experiments. cpm, counts per minute; *P < 0.05.

Fig. 2.

Measurement of endogenous aminotransferase activity with different amino donor and acceptor substrates. Activities were measured from isolated mitochondria in comparison with a fraction from density gradient purification containing chloroplasts and peroxisomes. Shown are the means ±SE from four independent experiments. AGA, alanine:glyoxylate aminotransferase activity; AKA, alanine:keto-glutarate aminotransferase activity; GGA, glutamate:glyoxylate aminotransferase activity; GPA, glutamate:pyruvate aminotransferase activity.

Fig. 3.

Subcellular localization of ALAAT1 and ALAAT2. Protoplasts were isolated from wild-type plants (A–C), plants overexpressing the ALAAT2–RFP fusion construct (D–F), and plants overexpressing the ALAAT1–RFP fusion construct (G–I). A, D, and G show transmitted light pictures (TLP) of the isolated protoplasts. B, E, and H show chlorophyll fluorescence (blue) and fluorescence of the MitoTracker® (green). C, F, and I show chlorophyll fluorescence in addition to RFP fluorescence (magenta). Images were acquired with a ×63 oil immersion PLAN-APO objective. The entire sample was excited with 488 nm and 568 nm laser light. The confocal sections were collected using a 515–535 nm emission setting for MitoTracker®GreenFM, 570–610 nm emission setting for RFP, and 660–720 nm emission setting for chlorophyll fluorescence.

Fig. 4.

Measurement of ALAAT2 aminotransferase activity with different substrates. Recombinant ALAAT2 was overexpressed in E. coli and activity was measured from crude extracts with different donor and acceptor substrates. Activites measured from a strain overexpressing an unrelated protein were substracted from those measured from ALAAT2 overexpressors. Shown are the means ±SE from three independent experiments. AGA, alanine:glyoxylate aminotransferase activity; AKA, alanine:ketoglutarate aminotransferase activity; GGA, glutamate:glyoxylate aminotransferase activity; GPA, glutamate:pyruvate aminotransferase activity.

Fig. 5.

Down-regulation of Alaat1 and Alaat2 expression by RNAi. Expression of Alaat1 and Alaat2 was measured in RNAi 1 and RNAi 1/2 lines (black bars) in comparison with wild-type expression (grey bars). Wild-type expression was set to 100%. Shown are the means ±SE from each 18 independent plants.

Fig. 6.

Alanine aminotransferase activity in wild-type (WT), Alaat1 knock-down plants (RNAi 1), and Alaat1/Alaat2 double knock-down plants (RNAi 1/2). (A) Alanine:glyoxylate aminotransferase activity. (B) Alanine:ketoglutarate aminotransferase. AOA was used as an aminotransferase inhibitor. Shown are the means ±SE from three independent experiments. *P < 0.05; ***P < 0.005.

Fig. 7.

Mitochondrial glycolate conversion in Arabidopsis RNAi lines and transiently overexpressing tobacco plants. Isolated mitochondria corresponding to 50 μg of protein were incubated with [¹⁴C]glycolate. ¹⁴CO₂ released was captured in NaOH and determined by scintillation counting. (A) ¹⁴CO₂ release in mitochondria from wild-type (WT), Alaat1 knock-down plants (RNAi 1), and Alaat1/Alaat2 double knock-down plants (RNAi 1/2). (B) ¹⁴CO₂ release in transiently transformed tobacco plants overexpressing red fluorescent protein (control), AlaAT1, GlcDH, or both AlaAT1 and GlcDH (AlaAT1+GlcDH). Shown are the means ±SE from at least three independent experiments. cpm, counts per minute; *P < 0.05; **P < 0.01.

Fig. 8.

Conservation of mitochondrial glycolate oxidation in rice. Glyoxylate formation was measured from isolated rice mitochondria. (A) GlcDH activity assay. Aminooxyacetate (AOA) or potassium cyanide (KCN) were added as inhibitors. (B) ¹⁴CO₂ release from isolated mitochondria with three different ¹⁴C-labelled substrates. AOA was added as an inhibitor. Values for all ¹⁴C-labelled substrates are corrected for the specific activity of each substrate. Shown are the means ±SE from at least three independent experiments. cpm, counts per minute; **P < 0.01; ***P < 0.005.