Fig 1.
The betalain biosynthetic pathway.
A simplified schematic representation of the main enzymatic and spontaneous reactions leading to the formation of red/purple betacyanins and yellow betaxanthins. Enzymatic steps: (1) tyrosine hydroxylation to ʟ-DOPA catalysed by CYP76AD cytochrome P450 enzymes; (2) cleavage of ʟ-DOPA to form betalamic acid by DODA; (3) ʟ-DOPA oxidation to cyclo-DOPA by CYP76AD1; and (4) cyclo-DOPA glycosylation to cyclo-DOPA 5-O-glucoside by the enzyme cDOPA5GT. S*, spontaneous reaction. Betacyanins are represented as a molecule of betanin. cDOPA5GT, cyclo-DOPA 5-O-glucosyltransferase; cyclo-DOPA, cyclo-dihydroxyphenylalanine; DODA, ʟ-DOPA-4,5-dioxygenase; ʟ-DOPA, 3,4-dihydroxy-ʟ-phenylalanine.
Fig 2.
Betalains can be produced in Medicago truncatula roots as a response to AM fungi colonisation.
(a) Schematic of the multigene vectors constructed for inducible betalain expression in M. truncatula roots where only the first gene of the betalain biosynthesis pathway is controlled by AM symbiosis–specific promoters. Expression of MtPT4-p1 (b, e, and h) and MtBCP1-p1 (c, f, and i) in roots of M. truncatula 4 weeks after inoculation with Rhizophagus irregularis. (d, g, and j) Example of MtPT4-p1 expressing root system mock inoculated with autoclaved R. irregularis. (e, f, g) Root system images filtered for red colouring only. Scale bar (b, c, and d), 1 cm; scale bar (h, i, and j), 1.5 mm. AM, arbuscular mycorrhiza.
Fig 3.
Betalain accumulation is observed at different intensities in different tissue layers and appears higher in the endodermal cells adjacent to arbuscule-containing cortical cells.
Root sections of Medicago truncatula expressing MtPT4-p1 (a and c) and MtBCP1-p1 (b and d) 4 weeks after inoculation with Rhizophagus irregularis. (c and d) Left: Betacyanin pigments are visible in red; right: WGA-FITC staining of fungal structures in blue. Open arrows mark internal hyphae, and filled arrows signal cells containing arbuscules. Scale bar, 100 μm. WGA, wheat germ agglutinin.
Fig 4.
Expression of NbPT5b and NbBCP1b genes is induced under colonisation with Rhizophagus irregularis in Nicotiana benthamiana.
(a) qRT-PCR analysis of NbPT5b and NbBCP1b shows that gene expression is induced 2 wpi with R. irregularis and increases with time and degree of colonisation. Y axes indicate expression levels relative to N. benthamiana’s elongation factor 1 alpha (NbEF). Expression of RiBTub is used as a fungal biomass marker for root colonisation. Raw qRT-PCR values can be found in S3 Data. (b and c) GUS staining of NbPT5b-GUS and NbBCP1b-GUS expressing N. benthamiana roots 4 weeks after inoculation with R. irregularis. GUS activity can only be observed in root areas colonised by R. irregularis and is more predominant in arbuscule-containing cells (white arrows). Scale bar, 500 μm. qRT-PCR, quantitative real-time polymerase chain reaction; RiBTub, R. irregularis β-tubulin; wpi, weeks postinoculation.
Fig 5.
Betalains can be produced in Nicotiana benthamiana roots as a response to AM fungi colonisation.
(a) Schematic of the multigene vectors constructed for inducible betalain expression in N. benthamiana roots where only the first gene of the betalain biosynthesis pathway is controlled by AM symbiosis–specific promoters. Expression of NbPT5b-p1 (b) and NbBCP1b-p1 (c) in whole root systems of N. benthamiana T1 plants 4 weeks after inoculation with Rhizophagus irregularis. Right: Root images are filtered for red colour. (d–g) Confocal microscopy of red NbPT5b-p1 (d and e) and NbBCP1b-p1 (f and g) transgenic root sections which were cut and stained with WGA-FITC to visualise fungal structures. (d and f) colour-filtered for red; (e and g) FITC green fluorescence. Scale bar (b and c), 1 cm; scale bar (d–g), 100 μm. AM, arbuscular mycorrhiza; WGA, wheat germ agglutinin.
Table 1.
Quantification of fungal structures observed in roots of Nicotiana benthamiana expressing NbPT5b-p1 and NbBCP1-p1 4 wpi with Rhizophagus irregularis.
Fig 6.
Betalain production in Nicotiana benthamiana roots as a response to AM fungi colonisation via expression of the entire betalain pathway under AM symbiosis–specific promoters.
(a) Schematic of the multigene vectors constructed for inducible betacyanin expression in N. benthamiana roots with the 3 betalain biosynthetic genes controlled by the NbPT5b or the NbBCP1 promoters. Expression of NbPT5b-p3 (b and c) and NbBCP1-p3 (d and e) in roots of N. benthamiana 4 weeks after inoculation with Rhizophagus irregularis. Betacyanin production was not visible in mock conditions for any NbPT5b-p3 or NbBCP1b-p3 roots. (c and e) are a magnification of the areas delimited by the dashed squares. Scale bar (b and d), 500 μm; scale bar (c and e), 100 μm. AM, arbuscular mycorrhiza; Ar, arbuscules; en, endodermis; ep, epidermis; ih, internal hyphae; v, vesicle.
Fig 7.
Red pigment distribution in root systems of NbBCP1b-p3 and NbPT5b-p3 Nicotiana benthamiana plants colonised by Rhizophagus irregularis.
Images were taken at 52 dpi. (a and c) are reflective light images, and (b, d, and e) are filtered for red colouring only. (e) High magnification image of (d) showing varying red colouration in parallel running roots. Scale bar = 1.3 cm. dpi, days postinoculation.
Fig 8.
NbBCP1b promoter-controlled betalain biosynthesis allows for dynamic tracing of root colonisation processes in Nicotiana benthamiana.
Transgenic NbBCP1-p3 plants grown in a rhizotron setup supplied with Rhizophagus irregularis spore inoculum imaged over time. (a) Reflective light images, (b) represent the same images filtered for red/magenta hues, and (c) is a magnification of the area delimited by the dashed square over time. Scale bar, 1 cm. dpi, days postinoculation.
Fig 9.
Betalains can be used as markers for AM colonisation in plant roots.
Left: Red pigmentation is easily observable in whole plant root systems. Right: Red pigmentation is most prominent in colonised tissues as well as in adjacent tissue layers.