The formation of Anthocyanic Vacuolar Inclusions in Arabidopsis thaliana and implications for the sequestration of anthocyanin pigments

Abstract

Anthocyanins are flavonoid pigments that accumulate in the large central vacuole of most plants. Inside the vacuole, anthocyanins can be found uniformly distributed or as part of sub-vacuolar pigment bodies, the Anthocyanic Vacuolar Inclusions (AVIs). Using Arabidopsis seedlings grown under anthocyanin-inductive conditions as a model to understand how AVIs are formed, we show here that the accumulation of AVIs strongly correlates with the formation of cyanidin 3-glucoside (C3G) and derivatives. Arabidopsis mutants that fail to glycosylate anthocyanidins at the 5-O position (5gt mutant) accumulate AVIs in almost every epidermal cell of the cotyledons, as compared to wild-type seedlings, where only a small fraction of the cells show AVIs. A similar phenomenon is observed when seedlings are treated with vanadate. Highlighting a role for autophagy in the formation of the AVIs, we show that various mutants that interfere with the autophagic process (atg mutants) display lower numbers of AVIs, in addition to a reduced accumulation of anthocyanins. Interestingly, vanadate increases the numbers of AVIs in the atg mutants, suggesting that several pathways might participate in AVI formation. Taken together, our results suggest novel mechanisms for the formation of sub-vacuolar compartments capable of accumulating anthocyanin pigments.

Keywords: Anthocyanin; autophagy; cyanidin 3-glucoside; vacuolar inclusion; vanadate.

Figure 1.

Anthocyanin Profiles of Arabidopsis Seedlings in the Presence or Absence of Naringenin. HPLC chromatographic profiles of anthocyanins at 520 nm of 3-day-old wild-type (Ler) (A, B); Col (C, D); and tt5 (E, F) grown in the absence (–Nar., A, C, and E) or presence (+Nar., B, D, and F) of 100 μM naringenin. Peaks ‘a, b, c, and d’ (red lines) correspond to anthocyanins newly accumulated upon the addition of naringenin. Peaks 1–13 correspond to anthocyanins previously described and characterized in Table 1. Anthocyanins were extracted from identical amounts of dry material, and the same volume was injected in each run.

Figure 2.

Time-Course Accumulation of Anthocyanin Peaks a, b, and c after Naringenin Addition. Quantification of anthocyanin peaks a, b, and c in 3-day-old wild-type (Ler) seedlings treated with 100 μM naringenin for different (0, 3, 6, 9, 12, and 24-h) periods of time. The quantification is based on peak area (μVol*sec) measurements of HPLC chromatograms revealed at 520 nm.

Figure 3.

Anthocyanin Accumulation in 3gt and 5gt Seedlings. (A) Spectrophotometric measurement (520 nm) of anthocyanin contents of 3gt, 5gt, and wild-type (Col) seedlings grown in AIC in the absence (–Nar) or presence (+Nar) of 100 μM naringenin. Anthocyanin accumulation levels correspond to arbitrary units obtained by dividing the absorbance at 520 nm by the mass of the starting plant material and the volume fraction used in the measurements. (B–D) Chromatographic profiles (520 nm) of 5gt and Col 3-day-old seedlings grown in the absence (A) or presence (B and C) of 100 μM naringenin.

Figure 4.

AVI Formation in 3gt and 5gt Seedlings. (A–F) Three-day-old 3gt and 5gt mutants and wild-type (Col) seedlings treated with 100 μM naringenin (+Nar), treated or not treated with 1 mM vanadate (Na₃VO₄, +/–Van). Scale bars: A–F: 0.5 mm. (G, H) Microscopic view of Col and 5gt mutant cells (adaxial side of the cotyledons), treated with 100 μM naringenin (+Nar). Scale bar G–H: 30 μm.

Figure 5.

Quantification of AVI and NRSB Numbers in 3gt, 5gt, and Col Seedlings Induced with Naringenin. (A) Number of AVIs and (B) number of neutral red staining sub-vacuolar structures (NRSBs) present in 5gt and wild-type (Col) seedlings treated with 100 μM naringenin (+Nar), treated or left untreated with 1 mM vanadate (+/–Van). Seedlings were observed 24 h after the addition of naringenin and vanadate.

Figure 6.

Effect of Vanadate on Anthocyanin Profiles. HPLC chromatograms of anthocyanins (520 nm) of 3-day-old wild-type (Col) or 5gt seedlings grown in the presence (+Nar) of 100 μM naringenin, with or without the addition of 1 mM vanadate (+/–Van).

Figure 7.

Autophagic Mutants and Anthocyanin Accumulation. (A) Spectrophotometric measurement (520 nm) of anthocyanin content of atg5, atg9, atg10, and wild-type (Col) seedlings 4 and 5 d after germination (DAG) grown in AIC. Anthocyanin accumulation levels correspond to arbitrary units obtained by dividing the absorbance at 520 nm by the mass of the starting plant material and the volume fraction used in the measurements. (B) Microscopic pictures of atg5, atg9, atg10, and wild-type (Col) seedlings 4 and 5 days after germination (DAG) grown in AIC.

Figure 8.

Autophagic Mutants and Anthocyanin Profiles. HPLC chromatograms of anthocyanins (520 nm) of atg5, atg9, atg10, and wild-type (Col) seedlings 5 DAG grown in AIC in the absence (left panel) or presence of 1 mM vanadate (Van).

Figure 9.

AVI and NRSB Formation in Autophagic Mutants. (A) Number of AVIs and (B) number of neutral red staining sub-vacuolar structures (NRSBs) present in agt mutants and wild-type (Col) seedlings 4 or 5 DAG treated with 100 μM naringenin (+Nar), treated or left untreated with 1 mM vanadate (Van). Seedlings were observed 24 h after the addition of naringenin and/or vanadate. Asterisks indicate a significant difference compared to the wild-type values (one asterisk: P < 0.1; two-sided t-test and two asterisks: P < 0.05; two-sided t-test).