Lysine decarboxylase catalyzes the first step of quinolizidine alkaloid biosynthesis and coevolved with alkaloid production in leguminosae

Abstract

Lysine decarboxylase (LDC) catalyzes the first-step in the biosynthetic pathway of quinolizidine alkaloids (QAs), which form a distinct, large family of plant alkaloids. A cDNA of lysine/ornithine decarboxylase (L/ODC) was isolated by differential transcript screening in QA-producing and nonproducing cultivars of Lupinus angustifolius. We also obtained L/ODC cDNAs from four other QA-producing plants, Sophora flavescens, Echinosophora koreensis, Thermopsis chinensis, and Baptisia australis. These L/ODCs form a phylogenetically distinct subclade in the family of plant ornithine decarboxylases. Recombinant L/ODCs from QA-producing plants preferentially or equally catalyzed the decarboxylation of L-lysine and L-ornithine. L. angustifolius L/ODC (La-L/ODC) was found to be localized in chloroplasts, as suggested by the transient expression of a fusion protein of La-L/ODC fused to the N terminus of green fluorescent protein in Arabidopsis thaliana. Transgenic tobacco (Nicotiana tabacum) suspension cells and hairy roots produced enhanced levels of cadaverine-derived alkaloids, and transgenic Arabidopsis plants expressing (La-L/ODC) produced enhanced levels of cadaverine, indicating the involvement of this enzyme in lysine decarboxylation to form cadaverine. Site-directed mutagenesis and protein modeling studies revealed a structural basis for preferential LDC activity, suggesting an evolutionary implication of L/ODC in the QA-producing plants.

Figure 1.

Biosynthetic Pathway of QAs. Biosynthetic pathway of QAs from l-Lys via cadaverine. LDC is responsible for the first step of biosynthesis.

Figure 2.

Phylogenetic Tree of Eukaryotic L/ODCs. Unrooted neighbor-joining phylogenetic tree of selected eukaryotic L/ODCs. The active-site amino acid residues that differ between LDC and ODC proteins are indicated for each sequence of the tree. Residue number is based on La-L/ODC numbering. Green boxes and red triangles indicate L/ODC and ODC enzymes, respectively, whose biochemical properties have been investigated. Magenta lines indicate quinolizidine alkaloid–producing plants. Bootstrap values (1000 replicates) are shown above each branch. Accession numbers of enzymes are listed in Supplemental Table 2 online.

Figure 3.

Comparison of the Human ODC (Hs-ODC) Structure with Predicted Protein Structures of La-L/ODC, La-L/ODC Mutant, and Ng-ODC with the Schiff Base Intermediate of Putrescine with PLP at the Active Site. Hs-ODC (A) was used as a template for protein modeling. Models are for La-L/ODC-WT (wild-type protein) (B), La-L/ODC-F344H (a site-directed mutant protein) (C), and Ng-ODC (D). The magenta arrows indicate the critical structural change regarding the extension of 3₁₀-helix, which could be responsible for accepting cadaverine and putrescine. The picture was created by PyMOL. Put, putrescine.

Figure 4.

Molecular Analysis of L. angustifolius L/ODC. (A) Accumulation of La-L/ODC mRNA in various tissues of two cultivars, bitter (cv Fest) and sweet (cv Uniharvest), of L. angustifolius plants by quantitative RT-PCR analysis. (B) Genomic PCR analysis of La-L/ODC. DNA was extracted from young leaves of both cultivars and subjected to genomic PCR analysis using the same primer pair that was used for quantitative RT-PCR analysis. Lane M, λPstI marker. (C) DNA gel blot analyses of La-L/ODC. Fifteen micrograms of genomic DNA of both cultivars was digested with EcoRI (lane I), EcoRV (lane II), and HindIII (lane III). The La-L/ODC ORF was used as a probe for hybridization. In both cultivars, at least one EcoRI- and HindIII-digested band was hybridized at 4.4 and 4.2 kb, respectively.

Figure 5.

Plastid Localization of L. angustifolius L/ODC N-Terminal 100 Amino Acids Fused with GFP in Arabidopsis Leaves. (A) Arabidopsis leaves expressing L. angustifolius L/ODC₁₀₀-GFP and Wx-TP-DsRed. L/ODC₁₀₀-GFP, green (left); Wx-TP-DsRed, red (middle); GFP and DsRed merged image (right). (B) Control experiments. Rubisco-TP-GFP (left), Wx-TP-DsRed (middle), and GFP and DsRed merged image (right). Bars = 10 μm.

Figure 6.

Overexpression of L. angustifolius L/ODC in Tobacco Hairy Roots and BY-2 Suspension Cells. Biosynthetic pathway of tobacco alkaloids with the average contents of tobacco alkaloids in La-L/ODC–overexpressing (LDC) hairy roots and BY-2 cells and compared with the corresponding levels in the control lines expressing a bacterial GUS gene. For hairy roots, each bar represents the mean ± se of average contents of tobacco alkaloids in six independent L/ODC-overexpressing lines (biological replicates; n = 4 for each line) and four independent GUS-expressing lines (biological replicates; n = 4 for each line). For tobacco BY-2 cells, each bar shows the mean ± se of average contents of tobacco alkaloids in eight independent L/ODC-overexpressing BY-2 cells and five independent control lines expressing GUS gene; for BY-2 cells, both cell lines were treated with methyl jasmonate. Student’s one-tailed t test, *P value < 0.05. Broken lines indicate unresolved reactions. DW, dry weight; FW, fresh weight.

Figure 7.

Overexpression of L. angustifolius L/ODC in Arabidopsis Plants. (A) The contents of cadaverine in wild-type and transgenic Arabidopsis plants expressing GUS or L/ODC (LDC). FW, fresh weight. (B) Quantitative RT-PCR analysis of La-L/ODC transcript in wild-type (WT), GUS-expressing (GUS), and La-L/ODC–overexpressing (LDC) Arabidopsis plants. (C) The average contents of Lys, Orn, cadaverine, and putrescine in wild-type and transgenic Arabidopsis plants expressing GUS or La-L/ODC (LDC). Expression of La-L/ODC was measured by quantitative RT-PCR, and the expression ratio was calculated based on the β-tubulin gene. For (A) and (B), each bar represents the mean ± se of four to six biological replicates for each independent line. For (C), each bar represents the mean ± se of the wild type (biological replicate; n = 4), six independent La-L/ODC–overexpressing lines (biological replicates; n = 4 to 6 for each line), and four independent GUS-expressing lines (biological replicates; n = 6 for each line). Student’s one-tailed t test, *P value < 0.05. ND, not detected.