The phosphorylated pathway of serine biosynthesis affects sperm, embryo, and sporophyte development, and metabolism in Marchantia polymorpha.

Serine metabolism is involved in various biological processes. Here we investigate primary functions of the phosphorylated pathway of serine biosynthesis in a non-vascular plant Marchantia polymorpha by analyzing knockout mutants of MpPGDH encoding 3-phosphoglycerate dehydrogenase in this pathway. Growth phenotypes indicate that serine from the phosphorylated pathway in the dark is crucial for thallus growth. Sperm development requires serine from the phosphorylated pathway, while egg formation does not. Functional MpPGDH in the maternal genome is necessary for embryo and sporophyte development. Under high CO2 where the glycolate pathway of serine biosynthesis is inhibited, suppressed thallus growth of the mutants is not fully recovered by exogenously-supplemented serine, suggesting the importance of serine homeostasis involving the phosphorylated and glycolate pathways. Metabolomic phenotypes indicate that the phosphorylated pathway mainly influences the tricarboxylic acid cycle, the amino acid and nucleotide metabolism, and lipid metabolism. These results indicate the importance of the phosphorylated pathway of serine biosynthesis in the dark, in the development of sperm, embryo, and sporophyte, and metabolism in M. polymorpha.

Identification and Biochemical Characterization of the Serine Biosynthetic Enzyme 3-Phosphoglycerate Dehydrogenase in Marchantia polymorpha.

L-serine is an important molecule in all living organisms, and thus its biosynthesis is considered to be regulated according to demand. 3-Phosphoglycerate dehydrogenase (PGDH), the first committed enzyme of the phosphorylated pathway of L-serine biosynthesis, is regulated by negative feedback from L-serine in bacteria. In the case of the vascular plant Arabidopsis thaliana, two PGDH isozymes out of three are inhibited by L-serine and activated by L-alanine, L-valine, L-methionine, L-homoserine, and L-homocysteine, suggesting a more complicated regulatory mechanism of L-serine biosynthesis in A. thaliana than in bacteria. However, it remains to be clarified whether the activation mechanism of PGDH by amino acids is conserved in land plants. In this study, we identified the sole isozyme of PGDH in the liverwort Marchantia polymorpha (MpPGDH) and elucidated its biochemical characteristics. MpPGDH cDNA encodes a 65.6 kDa protein that contains a putative transit peptide for chloroplast localization. MpPGDH shares 75-80% identity with A. thaliana isozymes and forms a homotetramer in vitro. Recombinant MpPGDH exhibited an optimal pH of 9.0, apparent Michaelis constants of 0.49 ± 0.04 and 0.096 ± 0.010 mM for 3-PGA and NAD+, respectively, and apparent maximum velocity of 5.65 ± 0.10 µmol⋅min-1⋅mg-1, similar to those of A. thaliana isozymes. Phosphate ions were found to stabilize MpPGDH, suggesting that phosphate ions are also a crucial factor in the regulation of serine biosynthesis via the phosphorylated pathway in Marchantia polymorpha. MpPGDH was inhibited by L-serine in a cooperative manner and was activated by L-alanine, L-valine, L-methionine, L-homoserine, and L-homocysteine to a lesser extent than it is in A. thaliana. The results suggest that an ancestral PGDH of land plants was inhibited byL-serine and slightly activated by five other amino acids.

Comparative metabolite profiling of foxglove aphids (Aulacorthum solani Kaltenbach) on leaves of resistant and susceptible soybean strains.

Aphid infestations can cause severe decreases in soybean (Glycine max [L.] Merr.) yield. Since planting aphid-resistant soybean strains is a promising approach for pest control, understanding the resistance mechanisms employed by aphids is of considerable importance. We compared aphid resistance in seven soybean strains and found that strain Tohoku149 was the most resistant to the foxglove aphid, Aulacorthum solani Kaltenbach. We subsequently analyzed the metabolite profiles of aphids cultured on the leaves of resistant and susceptible soybean strains using capillary electrophoresis-time-of-flight mass spectrometry. Our findings showed that the metabolite profiles of several amino acids, glucose 6-phosphate, and components of the tricarboxylic acid cycle were similar in aphids reared on Tohoku149 leaves and in aphids maintained under conditions of starvation, suggesting that Tohoku149 is more resistant to aphid feeding. Compared to susceptible strains, we also found that two methylated metabolites, S-methylmethionine and trigonelline, were either not detected or decreased in aphids reared on Tohoku149 plants. Since these metabolites function as important sulfur transporters in phloem sap and osmoprotectants involved in salt and drought stress, respectively, aphid-resistance is considered to be related to sulfur metabolism and methylation. These results contribute to an increase in our understanding of soybean aphid resistance mechanisms at the molecular level.

Metabolomic profiling of the response of susceptible and resistant soybean strains to foxglove aphid, Aulacorthum solani Kaltenbach.

Aphid infection reduces soybean (Glycine max [L.] Merr.) yield. Consequently, cultivation of aphid-resistant strains is a promising approach to pest control, and understanding the resistance mechanism is of importance. Here, we characterized the resistance of soybeans to foxglove aphid, Aulacorthum solani Kaltenbach, at the metabolite level. First, we evaluated aphid mortality and settlement rates on the leaves of two soybean strains, 'Tohoku149' and 'Suzuyutaka', and found that the former had strong resistance soon after introduction of the aphids. The metabolomic response to aphid introduction was analyzed using capillary electrophoresis-time-of-flight mass spectrometry. We found the following three features in the profiles: (1) concentrations of citrate, amino acids, and their intermediates were intrinsically higher for Tohoku149 than Suzuyutaka, (2) concentrations of several metabolites producing secondary metabolites, such as flavonoids and alkaloids, drastically changed 6h after aphid introduction, and (3) concentrations of TCA cycle metabolites increased in Tohoku149 48 h after aphid introduction. We also profiled free amino acids in aphids reared on both soybean strains and under starvation, and found that the profile of the aphids on Tohoku149 was similar to that of the starved aphids, but different to that of aphids on Suzuyutaka. These tests confirmed that aphids suck phloem sap even from Tohoku149. This study demonstrates the metabolomic profiles of both soybean strains and aphids, which will contribute to the molecular level understanding of mechanisms of soybean resistance to aphids.