Cytosolic and Nuclear Co-localization of Betalain Biosynthetic Enzymes in Tobacco Suggests that Betalains Are Synthesized in the Cytoplasm and/or Nucleus of Betalainic Plant Cells

Abstract

Betalains replace anthocyanins as color pigments in most families of Caryophyllales. Unlike anthocyanins, betalains are derived from tyrosine via three enzymatic steps: hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA; step 1), and conversion of L-DOPA to betalamic acid (step 2), and to cyclo-DOPA (cDOPA; step 3). The principal enzymes responsible for these reactions have been elucidated at the molecular level, but their subcellular localizations have not been explored; hence, the intracellular compartments wherein betalains are biosynthesized remain unknown. Here, we report on the subcellular localization of these principal enzymes. Bioinformatic predictors and N- and C-terminal GFP tagging in transgenic tobacco, showed that Beta vulgaris CYP76AD1 which mediates both steps 1 and 3, DODA1 that catalyzes step 2, and CYP76AD6 which also mediates step 1, were similarly localized to the cytoplasm and nucleus (although the P450s were also weakly present in the endoplasmic reticulum). These two compartments were also the principal locations of Mirabilis jalapa cDOPA5GT. The cytoplasmic and nuclear co-localization of these key enzymes in tobacco suggests that betalains are biosynthesized in the cytoplasm and/or nucleus of betalain-containing plant cells. Elucidation of the subcellular compartmentation of betalain biosynthesis will facilitate the bioengineering of the betalain biosynthetic pathway in non-betalain-containing plants.

Keywords: CYP76AD1; DODA1 (DOD); betalain biosynthesis; cDOPA5GT; cytoplasm; nucleus; subcellular compartment; subcellular localization.

FIGURE 1

The betalain biosynthetic pathway starting from tyrosine. Two enzymes, CYP76AD1 and DODA1 mediate all three major reactions for formation of betalain backbone. Solid lines, reactions approved by experiments at biochemical and molecular levels. Dashed lines, reactions proposed in the literatures. Spon., spontaneous reactions.

FIGURE 2

Confocal GFP fluorescence scanning of N-terminal tagged gene transgenic tobacco leaf cells. (A) GFP-CYP76AD1 and GFP-DODA1; (B) GFP-CYP76AD6 and GFP-cDOPA5GT. Every GFP construct and nucleolus-localized RFP marker or dual nucleus and cytoplasm RFP marker were co-transformed to tobacco (N. benthamiana) leaf epidermal cells by agro-infiltration, and 2 or 3 days post infiltration, the cells were assessed for fluorescence under Carl Zeiss 710 confocal laser scanning microscopy. For each enzyme, the upper panel shows red fluorescence from nucleolus marker RFP, the middle one, red fluorescence from chloroplast autofluorescence, and the lower one, dual nucleus and cytoplasm marker RFP. AD1, CYP76AD1; AD6, CYP76AD6; 5GT, cDOPA5GT; Nuc RFP, Nucleolus-localized RFP marker; Chl, Chloroplast autofluorescence; Nuc-cyt RFP, dual nucleus and cytoplasm RFP marker; GFP-Gene, GFP tagged at the N-terminus of the gene; Bar = 20 μm.

FIGURE 3

Confocal GFP fluorescence scanning of C-terminal tagged gene transgenic tobacco leaf cells. (A) CYP76AD1-GFP and DODA1-GFP; (B) CYP76AD6-GFP and -cDOPA5GT-GFP. The figure explanation is the same as that for Figure 2, except Gene-GFP, GFP tagged at the C-terminus of the gene.