PMC

Carotenoid-deficient plants have few prebranch sites. pDR5:LUC expression in control (A and B) and CPTA-treated seedlings (C and D). Luciferase activity (A and C) and overlays of luciferase activity and bright-field images (B and D) are shown. Color bars represent the range of analog-digital units (ADU) for luciferase activity. (Scale bars: 1 cm.) (E) Quantification of prebranch site number in control and CPTA-treated seedlings; the difference is statistically significant (Student t test, P < 1 × 10⁻⁶). Error bars represent SD. pDR5:LUC expression in the OZ is shown over time in control (F) and CPTA-treated (G) seedlings.

Treatment with the carotenoid cleavage inhibitor, D15, reduces LR capacity and prebranch site number without inducing an albino phenotype. pDR5:LUC expression in control (A, E, and F) and D15-treated (B, G, and H) seedlings. Quantification of LR capacity (C) and prebranch site number (D) are shown; these differences are statistically significant (Student t test, P < 1 × 10⁻⁶). Error bars represent SD. Luciferase activity (E and G) and luciferase activity overlaid with bright-field images (F and H) are shown. Color bars in E and G represent the range of ADU for luciferase activity. (Scale bar: 1 cm.)

Mutant analyses and ABA supplementation indicate that a β-carotene derivative distinct from ABA or strigolactone participates in LR formation. Quantification of LR capacity in mutants required for α-carotene biosynthesis (A) and mutations in each NCED and two ABA biosynthesis genes compared with their respective controls (B). For comparison, the change in LR capacity of CTPA-treated seedlings is included. (C) Quantification of LR capacity in control, 0.1 μM ABA, 0.5 μM ABA or CPTA, and 0.5 μM ABA-treated seedlings. (D) Quantification of prebranch site number in control and 0.5 μM ABA-treated seedlings [this difference is not statistically significant (Student t test, P > 0.05)]. (E and F) pDR5:LUC expression in seedlings grown on CPTA and 0.5 μM ABA. Luciferase activity (E) and overlay of bright-field and luciferase activity (F) are shown. Color bar in E represents range of ADU for luciferase activity. (Scale bar: 1 cm.) (G) Quantification of LR capacity in seedlings with mutations in CCD genes and MAX2 compared with their respective controls. Asterisks indicate statistically significant differences compared with controls (Student t test: *P < 1 × 10⁻³; **P < 1 × 10⁻⁶). Error bars represent SD.

Carotenoid-deficient seedlings produce few LRs. (A) Simplified version of the carotenoid biosynthesis pathway. Carotenoid- and apocarotenoid-specific biosynthesis steps are depicted in black, and upstream steps are shown in dark gray. Carotenoid biosynthesis mutants used in this study are indicated (gray italics), and those with an indirect impact are shown in parentheses. The dotted arrow to strigolactone represents unknown final steps. Columbia-0 (Col-0) seedlings were grown under control (B) and CPTA treatment (C) conditions. (D) Quantification of LR capacity in control and NF- or CPTA-treated seedlings. (E) Col-0 and ispH1/clb6, psy, and sca3 albino seedlings. (F) Quantification of LR capacity in Col-0 and albino seedlings. The difference between control and treated or control and mutant in D and F, respectively, is statistically significant (Student t test, P < 1 × 10⁻⁶). (G) CPTA-treated seedlings transferred to control medium (yellow dotted line indicates root tip position at transfer). Arrowheads indicate emerged LRs: those formed in the presence of CPTA (white) and those formed after transfer (yellow). Error bars represent SD. (Scale bars: 1 cm.)

Expression of carotenoid biosynthesis genes is consistent with a role for carotenoids in LR formation. (A) Heat map of mean expression values of the core carotenoid biosynthesis genes in the root cell types (). Core carotenoid biosynthesis gene expression is higher and most similar among cell types closely associated with LR formation (red). Note these genes are listed by their position in the pathway with PSY at the top (). C cells, companion cells; Devel. Phloem, developing phloem; Epid-hair, epidermis hair cells; Epid-nonhair, epidermis nonhair cells; LRC, lateral root cap; QC, quiescent center; P-M phloem, proto- and metaphloem; P-M xylem, proto- and metaxylem; PP Pericycle, phloem pole pericycle; XP pericycle, xylem pole pericycle. White boxes indicate mean expression values of <1.0. (B–G) pPSY:LUC reporter gene expression in roots. (B) Luciferase activity. (C) Overlay of luciferase activity from B and bright-field image. (D and E) Histograms of luciferase activity from B; red represents high values, and blue represents no expression. The bracket in D indicates the region shown in E. (E) Two-dimensional representation of D with LUC activity on the y axis and with the root along the x axis (root tip at right). (F) Luciferase activity at the base of LRs and in later stage primordia. (G) Overlay of luciferase activity from F and bright-field image. Color bars represent the range of ADU for luciferase activity. (Scale bars: B, C, and E, 10 mm; F and G, 1 mm.)