An L,L-diaminopimelate aminotransferase mutation leads to metabolic shifts and growth inhibition in Arabidopsis

Abstract

Lysine (Lys) connects the mitochondrial electron transport chain to amino acid catabolism and the tricarboxylic acid cycle. However, our understanding of how a deficiency in Lys biosynthesis impacts plant metabolism and growth remains limited. Here, we used a previously characterized Arabidopsis mutant (dapat) with reduced activity of the Lys biosynthesis enzyme L,L-diaminopimelate aminotransferase to investigate the physiological and metabolic impacts of impaired Lys biosynthesis. Despite displaying similar stomatal conductance and internal CO2 concentration, we observed reduced photosynthesis and growth in the dapat mutant. Surprisingly, whilst we did not find differences in dark respiration between genotypes, a lower storage and consumption of starch and sugars was observed in dapat plants. We found higher protein turnover but no differences in total amino acids during a diurnal cycle in dapat plants. Transcriptional and two-dimensional (isoelectric focalization/SDS-PAGE) proteome analyses revealed alterations in the abundance of several transcripts and proteins associated with photosynthesis and photorespiration coupled with a high glycine/serine ratio and increased levels of stress-responsive amino acids. Taken together, our findings demonstrate that biochemical alterations rather than stomatal limitations are responsible for the decreased photosynthesis and growth of the dapat mutant, which we hypothesize mimics stress conditions associated with impairments in the Lys biosynthesis pathway.

Fig. 1.

Schematic representation of lysine turnover (biosynthesis and degradation). Lysine is synthetized in the chloroplast using aspartate as a precursor. Dihydrodipicolinate synthase (DHDPS) is the first enzyme of lysine biosynthesis and it requires pyruvate export from the cytosol to the chloroplast. Under stress conditions, lysine is exported from the chloroplast to mitochondria to be degraded (trace arrows), and electrons are used as a donor for ATP synthesis in two ways: (i) lysine can be degraded by lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) resulting in acetyl-CoA entering in the TCA cycle, or (ii) lysine can be degraded by D-2-hydroxyglutarate dehydrogenase (D2HGDH) resulting in 2-oxoglutarate (2-OG), which at the same time acts as an electron donor for the alternative respiration system mediated by electron transfer flavoprotein (ETF)–electron transfer flavoprotein:ubiquinone oxidoreductase (ETFQO). Thus there is a close relationship between chloroplasts and mitochondria in lysine metabolism.

Fig. 2.

Effects of lysine biosynthesis deficiency observed in dapat mutant plants. (A) L,L-Diaminopimelate aminotransferase (DAPAT) enzyme activity showing drastic reduction in mutant plants. (B) Arabidopsis plants grown in short day conditions as described in ‘Materials and methods’. (C) WT and dapat seed germination assay showing a delay in germination of dapat seeds. (D) Arabidopsis seedling establishment of WT and dapat 5 d after start of germination. While WT showed germination of all seeds, a few dapat seeds were unable to germinate.

Fig. 3.

Changes in gas-exchange measurements in wild-type (WT) and mutant (dapat). (A) Photosynthesis. (B) Stomatal conductance to water vapor. (C) Internal CO₂ concentration. (D) Dark respiration. Bars are means ±SE from five biological replicates; *significantly different (P<0.01) from WT by Student’s t-test.

Fig. 4.

Effect of deficiency of lysine biosynthesis along diurnal cycle on starch (A), sucrose (B), glucose (C), fructose (D), malate (E), and fumarate (F) level. ED, end of day; EN, end of night. Values are means ±SE of five independent biological replicates. Asterisks designate values that were significantly different from WT (*P<0.05, **P<0.01) by Student’s t-test.

Fig. 5.

Effect of deficiency on lysine biosynthesis along a diurnal cycle on the total protein (A) and amino acid level (B). Values are means ±SE of five independent biological replicates. Asterisks designate values that were significantly different from WT (*P<0.05, **P<0.01) by Student’s t-test.

Fig. 6.

MapMan metabolic overview of the dapat mutant when compared with its wild-type correspondent. MapMan was loaded with 3138 transcripts that switched their expression at least 1.5-fold and showed ANOVA to P<0.05 into Arabidopsis major metabolic pathways. The log2 ratio values (dapat/WT) were plotted onto boxes in which up-regulated and down-regulated genes are indicated by the shading in accordance with the scale shown at the upper right.

Fig. 7.

Gene expression in dapat mutant. (A) Heatmap of catabolic genes retrieved from dapat transcriptome. The log2 ratio (dapat/WT) values were plotted onto boxes in which up-regulated and down-regulated genes are indicated by shading in accordance with the scale shown at the lower left. (B) validation of trancriptome by quantitative PCR of amino acid catabolism genes along diurnal cycle. *Significantly different (P<0.05) from WT within each time point by Student’s t-test.

Fig. 8.

Metabolic profiling of dapat mutant along diurnal cycle. ED, end of day; EN, end of night. Metabolites were determined as described in ‘Materials and methods’. The full datasets from these metabolic profiling studies are additionally available in Supplementary Table S4. The colour code of the heat map is given as the log₂ scale. Data are normalized with respect to the mean response calculated for WT (to allow statistical assessment, individual plants from this set were normalized in the same way). Values are means ±SE of four independent biological replicates. *Significantly different from WT (P<0.05) by Student’s t-test.

Fig. 9.

Schematic representation of metabolic reprogramming caused by the DAPAT mutation during diurnal cycle. Despite lower stomatal conductance, biochemical changes were responsible for lower photosynthesis, including an impact on carbohydrate metabolism resulting in putative energy deprivation and further accumulation of protein and amino acids, leading to C/N imbalance, which seems to be associated with the dwarfism phenotype in dapat plants. In order to obtain a metabolic adjustment, we postulate a hypothetical model in which malate is hyper-accumulated at the end of day (ED). Malate is further oxidized during the night by an alternative pathway associated with the cytosolic malic enzyme 2 to generated pyruvate and, thus, to sustain mitochondrial dark respiration at similar level when compared with WT plants. Furthermore, higher turnover of both protein and amino acids in dapat plants acts as a source for alternative respiration mediated by the ETF–ETFQO complex and dehydrogenases such as IVDH and D2HGDH. Data obtained from the transcriptome, proteome, and metabolome were integrated to build the mechanism described here. Rectangles represent transcript data; circles represent protein data, and underlined names represent metabolite data. Increased and reduced levels from the data obtained are shown in black uppercase and grey lowercase letters, respectively. Abbreviations: Cb6f, complex b₆/f; CS, citrate synthase; cME2, cytosolic malic enzyme 2; D2HGDH, D-2-hydroxyglutarate dehydrogenase; DAPAT, L-L-diaminopimelate aminotransferase; ETF–ETFQO, electron-transfer flavoprotein–electron-transfer flavoprotein: ubiquinone oxidoredutase; GAD1, glutamate decarboxylase; GADPH, glyceraldehyde-3-phosphate dehydrogenase; IVDH, isovaleryl-CoA dehydrogenase; PRK, phosphoribulokinase; PSI, photosystem I; PSII, photosystem 2; TRX, thioredoxin; WT, wild type.