Biosynthesis and Metabolic Fate of Phenylalanine in Conifers

Abstract

The amino acid phenylalanine (Phe) is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development, and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of phenylpropanoids. The study of this metabolic pathway is particularly relevant in trees, which divert large amounts of carbon into the biosynthesis of Phe-derived compounds, particularly lignin, an important constituent of wood. The trunks of trees are metabolic sinks that consume a considerable percentage of carbon and energy from photosynthesis, and carbon is finally immobilized in wood. This paper reviews recent advances in the biosynthesis and metabolic utilization of Phe in conifer trees. Two alternative routes have been identified: the ancient phenylpyruvate pathway that is present in microorganisms, and the arogenate pathway that possibly evolved later during plant evolution. Additionally, an efficient nitrogen recycling mechanism is required to maintain sustained growth during xylem formation. The relevance of phenylalanine metabolic pathways in wood formation, the biotic interactions, and ultraviolet protection is discussed. The genetic manipulation and transcriptional regulation of the pathways are also outlined.

Keywords: aromatic amino acids; gene regulatory networks; nitrogen recycling; phenylpropanoids; trees.

FIGURE 1

General pathway for aromatic amino acid biosynthesis and derived products. The shikimate pathway connects central carbon metabolism (glycolysis and pentose phosphate pathways) with the biosynthesis of aromatic amino acids and derived products. PEP, phosphoenolpyruvate; E4P, erythrose 4-phosphate; CM, chorismate mutase; AS, anthranilate synthase.

FIGURE 2

The two alternative pathways for Phe biosynthesis and associated N recycling. PAT, prephenate aminotransferase; PDT, prephenate dehydratase; ADT, arogenate dehydratase; PPY-AT, phenylpyruvate aminotransferase; PAL, phenylalanine ammonia lyase; GS1b, glutamine synthetase 1b; NADH-GOGAT, NADH-dependent glutamate synthase; L-Glu, L-glutamate; L-Gln, L-glutamine; 2-OG, 2-oxoglutarate.

FIGURE 3

Simplified scheme of monolignol synthesis pathway in conifers. Thick arrows highlight the route channeling the higher amount of carbon in conifers, the synthesis of coniferyl alcohol. The gray and discontinued arrows highlight the sinapyl alcohol synthesis that does occur in conifers because of the lack a gene encoding the F5H enzyme. Asterisk highlights the lack of F5H enzyme in conifers. Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), cinnamoyl-CoA reductase (CCR), cinnamyl-alcohol dehydrogenase (CAD), shikimate O-hydroxycinnamoyltransferase (HCT), p-coumarate 3-hydroxylase (C3H), caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulic acid 5-hydroxylase/coniferaldehyde 5-hydroxylase (F5H), and caffeic acid 3-O-methyltransferase (COMT).

FIGURE 4

Phylogenetic tree of the deduced protein sequences of plant genes encoding caffeic acid 3-O-methyltransferase (COMT). In the tree, the protein sequences correspond to virtually all the sequences of O-methyltransferases from Arabidopsis thaliana, all the sequences of O-methyltransferases, type COMT, found in the Pinus pinaster SustainPineDB and the most similar sequence to Ath-COMT1 from the rest of the shown species. The CLUSTALW program was used for sequence alignments (Thompson et al., 1994). The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987) with 1000 bootstrap replications. The optimal tree with the sum of branch length = 12.56422456 is shown. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling, 1965) and are in the units of the number of amino acid substitutions per site. The analysis involved 60 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 156 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). The sequences used for the alignments and phylogenetic trees were obtained in Phytozome database (http://phytozome.jgi.doe.gov) and GenBank and, for P. pinaster, in SustainPineDB (http://www.scbi.uma.es/sustainpinedb/). The alignment, tree and accession numbers are available in TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S18814). Red triangles correspond to A. thaliana sequences and the blue circles to P. pinaster sequences. Purple branches correspond to the Ath-CCoAOMT group of sequences, red branches to the angiosperm COMT group and blue branches to the gymnosperm COMT-like group.