Research ArticleDEVELOPMENTAL BIOLOGY

Anchorene is a carotenoid-derived regulatory metabolite required for anchor root formation in Arabidopsis

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Science Advances  27 Nov 2019:
Vol. 5, no. 11, eaaw6787
DOI: 10.1126/sciadv.aaw6787
  • Fig. 1 Anchorene promotes ANR formation.

    (A) Proposed production of anchorene (AR) (12,12′-diapocarotene-12,12′-dial) from β-carotene by oxidative cleavage; all-trans-β-carotene is taken as an example for the C-atom numbering. (B) AR promotes ANR formation in a concentration-dependent manner. Bottom: Representative seedlings treated with the indicated AR concentration. Scale bar, 1 cm. Top: Quantification of ANR no. as percentage (means ± SD) of seedlings with zero, one, or two ANRs (three independent replicates) corresponding to the treatments in the bottom panel. UC, uncut root apical meristems (RAMs); C, cut RAMs. (C) Two opposing ANRs are formed at the collet. White and red arrows in the right panel indicate primary ANRs and secondary ANRs (or LRs), respectively. (D) ANRs are initiated from the pericycle cells of the root. pDR5::nls-YFP was used for confocal microscopy imaging. The yellow fluorescent protein (YFP) signal is indicated by green, and SCRI Renaissance 2200 staining is indicated by purple. Ep, epidermis; C1, cortex layer 1; C2, cortex layer 2; En, endodermis; P, pericycle; ARI, ANR initiation site; ARP, ANR primordia; RH, root hair. Photo credit: K.-P.J. and S.A.-B., KAUST (B and C) and T.T.X. and I.B., KAUST (D).

  • Fig. 2 Carotenoid deficiency leads to reduced ANR formation.

    (A) Schematic of plant carotenoid biosynthesis. The gray dotted arrow indicates the upstream steps; mutants are shown in gray italics; reactions inhibited by NF and CPTA are depicted in red. Representative seedlings (B) and quantification data (C) show the effect of NF, CPTA, and D15 treatment on ANRs and LRs formation in Col-0 seedlings. ANRs and LRs no. were quantified after RAM excision; data are presented as means ± SD (three independent replicates). *P < 0.05, **P < 0.01, and ***P < 0.001, by two-tailed paired Student’s t tests. (D) Comparison of ANR formation between Col-0 and psy and ispH1 mutants. (E) ANR-RE quantification in Col-0, psy, and ispH1 seedlings. Data are presented as means ± SD (three independent replicates); ***P < 0.001, by two-tailed paired Student’s t test. Representative 5 dps NF-treated pWOX5::GFP (F) or psy mutant (G) seedlings have initiated ANR primordium. Photo credit: K.-P.J. and S.A.-B., KAUST (B and D) and A.J.D. and P.N.B., Duke University (F and G).

  • Fig. 3 AR is an endogenous metabolite.

    Representative seedlings (A) or ANR-RE quantification (B) of NF- and CPTA-treated seedlings exposed to AR. Representative seedlings (C) or ANR-RE quantification (D) of psy mutant seedlings treated with AR. “−” and “+” in (A) and (C) indicate the absence or presence of AR; in (B) and (D), data are presented as the percentage (means ± SD) of seedlings with zero, one, or two ANRs (three independent replicates). NF (1 μM), CPTA (100 μM), and AR (20 μM) were used. Scale bars, 1 cm. (E) Liquid chromatography mass spectrometry (LCMS) identification of endogenous AR in Arabidopsis root extract. Extracted ion chromatograms of AR from Arabidopsis root extract (top), AR standard (AS; middle), and Arabidopsis root extract spiked with AS (bottom). Peak II indicates endogenous AR (top) or endogenous AR spiked with AS (bottom). Peaks I and III represent AR isomers. (F) Endogenous AR (peak II) from Arabidopsis root extract and AS displayed identical pattern of product ion spectra. m/z, mass/charge ratio. Photo credit: K.-P.J. and S.A.-B., KAUST (A and C).

  • Fig. 4 AR promotes ANR formation by regulating auxin distribution.

    (A) Effect of AR on ANR-RE in Col-0 and arf7arf19 seedlings. (B) Confocal microscopy imaging of the collet region in a representative arf7arf19 seedling. (C) Effect of NAA and NPA on ANR-RE. (D) AR partially rescued ANR-RE in NPA-treated seedlings. (E) A representative confocal microscopy image of the collet region to show the AR effect on pLAX3::LAX3-YFP expression during ANR primordia formation. The YFP channel is shown in green, and the SCRI Renaissance 2200 staining is shown in purple. ANR quantification (F) and a representative picture (G) to show the seedlings of Col-0 and lax3 under normal (mock), RAM excision (RE), and AR application conditions. In (A), (C), and (F), data are presented as means ± SEM from one representative experiment. In (A) (n = 49, 51, 44, and 50), different letters denote significant differences (one-way ANOVA with Tukey’s multiple comparisons test, P < 0.05). In (C) (n = 25, 27, 31, 28, 25, and 27) and (F) (n = 43, 44, 39, 45, 43, and 48), ***P < 0.001 by two-tailed Student’s t test. In (D), data are presented as means ± SD (three independent replicates); **P < 0.01, by two-tailed paired Student’s t test. Photo credit: A.J.D. and P.N.B., Duke University (B); T.T.X. and I.B., KAUST (E); and K.-P.J. and S.A.-B., KAUST (G).

  • Fig. 5 AR and ANR formation are triggered by nitrogen deficiency.

    (A) Representative 8-day-old sand- or soil-grown seedlings. White and red arrows indicate collets and ANRs, respectively. (B) ANR formation rate of seedlings grown in sand and soil. Representative seedlings (C) and ANR no. counting (D) of 10-dps Col-0 seedlings grown in agar plates with Hoagland (mock), phosphorus deficiency (−P), or nitrogen deficiency (−N) media. (E) AR contents of the root tissues of seedlings grown in the plates with Hoagland or −N media. (F) AR treatment increases root and shoot biomass of Arabidopsis seedlings. Seventeen-day-old seedlings with or without AR treatment were used for biomass analysis. In (B), (D), and (F), data are presented as means ± SD from three, four, and three independent experiments, respectively, and paired two-tailed Student’s t test was used; in (E), data are presented as means ± SEM from one representative experiment, and unpaired two-tailed Student’s t test was used (n = 6 and 3). *P < 0.05, **P < 0.01, and ***P < 0.001. Photo credit: K.-P.J. and S.A.-B., KAUST (A and C).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaaw6787/DC1

    Supplementary Text

    Fig. S1. Structures of diapocarotenoids and supposed precursors for anchorene.

    Fig. S2. Effects of diapocarotenoid and its derivatives on Arabidopsis root development.

    Fig. S3. Characterization of ANR development by different DR5 marker lines.

    Fig. S4. Root formation under different treatments and in various mutants.

    Fig. S5. Anchorene effects on DR5 expression in ANR primordia.

    Fig. S6. Anchorene isomer identification and anchorene quantification.

    Fig. S7. Conversion of OH-Apo12′ into anchorene in plants.

    Fig. S8. Involvement of auxin signaling and distribution on ANR development.

    Fig. S9. Transcriptomic change analysis of collet tissues upon different treatments by RNA-seq.

    Fig. S10. Effect of anchorene on plant growth.

    Fig. S11. The synthesis route and derivatization for anchorene.

    Table S1. The nutrient element composition in Argo soil and Silver sand.

    Table S2. Mutants and marker lines used in this study.

    Dataset S1. Gene list (1.5-fold change) for different treatments in RNA-seq.

    Dataset S2. BP enrichment for different treatments in RNA-seq.

    References (6164)

  • Supplementary Materials

    The PDFset includes:

    • Supplementary Text
    • Fig. S1. Structures of diapocarotenoids and supposed precursors for anchorene.
    • Fig. S2. Effects of diapocarotenoid and its derivatives on Arabidopsis root development.
    • Fig. S3. Characterization of ANR development by different DR5 marker lines.
    • Fig. S4. Root formation under different treatments and in various mutants.
    • Fig. S5. Anchorene effects on DR5 expression in ANR primordia.
    • Fig. S6. Anchorene isomer identification and anchorene quantification.
    • Fig. S7. Conversion of OH-Apo12′ into anchorene in plants.
    • Fig. S8. Involvement of auxin signaling and distribution on ANR development.
    • Fig. S9. Transcriptomic change analysis of collet tissues upon different treatments by RNA-seq.
    • Fig. S10. Effect of anchorene on plant growth.
    • Fig. S11. The synthesis route and derivatization for anchorene.
    • Table S1. The nutrient element composition in Argo soil and Silver sand.
    • Table S2. Mutants and marker lines used in this study.
    • Legends for datasets S1 and S2
    • References (6164)

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    Other Supplementary Material for this manuscript includes the following:

    • Dataset S1 (Microsoft Excel format). Gene list (1.5-fold change) for different treatments in RNA-seq.
    • Dataset S2 (Microsoft Excel format). BP enrichment for different treatments in RNA-seq.

    Files in this Data Supplement:

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