Additional betalain accumulation by genetic engineering leads to a novel flower color in lisianthus (Eustoma grandiflorum)

Abstract

Betalains, comprising violet betacyanins and yellow betaxanthins, are pigments found in plants belonging to the order Caryophyllales. In this study, we induced the accumulation of betalains in ornamental lisianthus (Eustoma grandiflorum) by genetic engineering. Three betalain biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in lisianthus, in which anthocyanin pigments are responsible for the pink flower color. During the selection process on hygromycin-containing media, some shoots with red leaves were obtained. However, most red-colored shoots were suppressed root induction and incapable of further growth. Only clone #1 successfully acclimatized and bloomed, producing pinkish-red flowers, with a slightly greater intensity of red color than that in wild-type flowers. T₁ plants derived from clone #1 segregated into five typical flower color phenotypes: wine red, bright pink, pale pink, pale yellow, and salmon pink. Among these, line #1-1 showed high expression levels of all three transgenes and exhibited a novel wine-red flower color. In the flower petals of line #1-1, abundant betacyanins and low-level betaxanthins were coexistent with anthocyanins. In other lines, differences in the relative accumulation of betalain and anthocyanin pigments resulted in flower color variations, as described above. Thus, this study is the first to successfully produce novel flower color varieties in ornamental plants by controlling betalain accumulation through genetic engineering.

Keywords: anthocyanins; betacyanins; flower color; lisianthus; transgenic plants.

Figure 1. Betalain biosynthetic pathway. CYP76AD1 exhibits tyrosine hydroxylase and L-DOPA oxygenase activity. DOPA 4,5-dioxygenase (DOD) converts L-DOPA into betalamic acid. Cyclo-DOPA 5-O-glucosyltransferase (5GT) catalyzes the glycosylation of cyclo-DOPA. Betacyanins are formed by the spontaneous conjugation of betalamic acid and cyclo-DOPA 5-O-glucoside. Betaxanthins are formed by the spontaneous conjugation of betalamic acid and amines or amino acids. Filled and empty arrows indicate enzymatic and spontaneous steps, respectively.

Figure 2. Vector constructs used in this study. A pShyg-35SpintronGUS (vector control). B pShyg-35SpCYP76AD1-35SpDOD-35Sp5GT. LB, left border; NosP, nopaline synthase promoter; HPT, hygromycin phosphotransferase; rbcT, Arabidopsis rubisco small subunit terminator; 35SP, cauliflower mosaic virus 35S promoter; GUS, β-glucoronidase with intron region; HT, Arabidopsis heat shock protein 18.2 terminator; RB, right border; CYP76AD1, Beta vulgaris CYP76AD1; DOD, Mirabilis jalapa DOPA 4,5-dioxygenase; 5GT, M. jalapa cyclo-DOPA 5-O-glucosyltransferase; H, Hind III restriction site.

Figure 3. Phenotypes of primary transgenic lisianthus plants expressing CYP76AD1, DOD, and 5GT. A Hygromycin-resistant shoots harboring intronGUS (left) and CYP76AD1-DOD-5GT (right). B Betalain-accumulating transgenic plantlets acclimated in soil. C Blooming flowers of the vector control line (intronGUS, left) and the CYP76AD1-DOD-5GT transgenic line (clone A; right). D T₁ seedlings of CYP76AD1-DOD-5GT transgenic lisianthus clone #1 at the seedling stage. Scale bar=2 cm.

Figure 4. Five typical flower color phenotypes of T₁ lines. VC, vector control T₁ line; lines #1-1 to #1-5, T₁ lines of CYP76AD1-DOD-5GT-expressing lisianthus clone #1. Scale bar=2 cm.

Figure 5. Accumulation of betacyanins, anthocyanins, and flavonols in the petals of T₁ lines. Petal extracts of each clone were subjected to high-performance liquid chromatography (HPLC) analyses, and betacyanins, anthocyanins, and flavonols were monitored at 538, 530, and 360 nm, respectively. The level of each pigment was exhibited as the peak area of chromatography. VC, vector control; #1-1 to #1-5, CYP76AD1-DOD-5GT-expressing T₁ lines. Data represent the mean±standard error (SE; n=3). Different letters above the columns indicate significant differences (p<0.05; Tukey–Kramer test).

Figure 6. Expression of transgenes and endogenous anthocyanin biosynthetic genes in the petals of T₁ lines. Semi-quantitative RT-PCR analyses of three transgenes (CYP76AD1, DOD, and 5GT), two endogenous anthocyanin biosynthetic genes (CHS and DFR), and an internal reference gene (ACT). Gene names are indicated on the left of each panel, and PCR cycle numbers are indicated on the right. Approximately 130, 190, 120, 310, 340, and 200 bp of CYP76AD1, DOD, 5GT, CHS, DFR, and ACT, respectively, were amplified.