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. 2017 Jul 11;17(1):121.
doi: 10.1186/s12870-017-1071-x.

Indole-3-butyric Acid Promotes Adventitious Rooting in Arabidopsis Thaliana Thin Cell Layers by Conversion Into indole-3-acetic Acid and Stimulation of Anthranilate Synthase Activity

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Free PMC article

Indole-3-butyric Acid Promotes Adventitious Rooting in Arabidopsis Thaliana Thin Cell Layers by Conversion Into indole-3-acetic Acid and Stimulation of Anthranilate Synthase Activity

L Fattorini et al. BMC Plant Biol. .
Free PMC article

Abstract

Background: Indole-3-acetic acid (IAA), and its precursor indole-3-butyric acid (IBA), control adventitious root (AR) formation in planta. Adventitious roots are also crucial for propagation via cuttings. However, IBA role(s) is/are still far to be elucidated. In Arabidopsis thaliana stem cuttings, 10 μM IBA is more AR-inductive than 10 μM IAA, and, in thin cell layers (TCLs), IBA induces ARs when combined with 0.1 μM kinetin (Kin). It is unknown whether arabidopsis TCLs produce ARs under IBA alone (10 μM) or IAA alone (10 μM), and whether they contain endogenous IAA/IBA at culture onset, possibly interfering with the exogenous IBA/IAA input. Moreover, it is unknown whether an IBA-to-IAA conversion is active in TCLs, and positively affects AR formation, possibly through the activity of the nitric oxide (NO) deriving from the conversion process.

Results: Revealed undetectable levels of both auxins at culture onset, showing that arabidopsis TCLs were optimal for investigating AR-formation under the total control of exogenous auxins. The AR-response of TCLs from various ecotypes, transgenic lines and knockout mutants was analyzed under different treatments. It was shown that ARs are better induced by IBA than IAA and IBA + Kin. IBA induced IAA-efflux (PIN1) and IAA-influx (AUX1/LAX3) genes, IAA-influx carriers activities, and expression of ANTHRANILATE SYNTHASE -alpha1 (ASA1), a gene involved in IAA-biosynthesis. ASA1 and ANTHRANILATE SYNTHASE -beta1 (ASB1), the other subunit of the same enzyme, positively affected AR-formation in the presence of exogenous IBA, because the AR-response in the TCLs of their mutant wei2wei7 was highly reduced. The AR-response of IBA-treated TCLs from ech2ibr10 mutant, blocked into IBA-to-IAA-conversion, was also strongly reduced. Nitric oxide, an IAA downstream signal and a by-product of IBA-to-IAA conversion, was early detected in IAA- and IBA-treated TCLs, but at higher levels in the latter explants.

Conclusions: Altogether, results showed that IBA induced AR-formation by conversion into IAA involving NO activity, and by a positive action on IAA-transport and ASA1/ASB1-mediated IAA-biosynthesis. Results are important for applications aimed to overcome rooting recalcitrance in species of economic value, but mainly for helping to understand IBA involvement in the natural process of adventitious rooting.

Keywords: Adventitious roots; Anthranilate synthase genes; In vitro culture; Indole-3-acetic acid; Indole-3-acetic acid efflux carriers; Indole-3-acetic acid influx carriers; Indole-3-butyric acid; Nitric oxide; Stem thin cell layers; ech2ibr10 mutant.

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Figures

Fig. 1
Fig. 1
Adventitious rooting in Arabidopsis thaliana TCLs under different hormonal treatments. a Percentage of TCLs, Col-0 ecotype, either at the initial stage or with macroscopic callus and ARs, after 15 days of culture without hormones (HF) or with IBA (10 μM) + Kin (0.1 μM), IBA (10 μM), IAA (10 μM) or Kin (0.1 μM). b Productivity of AR-forming TCLs evaluated as mean number (±SE) of ARs per TCL under either IBA (10 μM) + Kin (0.1 μM), or IBA (10 μM), or IAA (10 μM). a,d P < 0.01 difference with respect to the other treatments within the same developmental stage. b,c, P < 0.01 difference with respect to IBA + Kin and IBA within the same developmental stage. e, P < 0.01 difference with respect to IAA. f, P < 0.001 difference with respect to IBA + Kin. Columns with no letter or the same letter within the same developmental stage are not significantly different. N = 100
Fig. 2
Fig. 2
Macroscopic adventitious rooting response on TCLs from various genotypes under different hormonal treatments. ag Images under the stereomicroscope at the end of the in vitro culture (day 15) with IBA (10 μM) (a, d, f, g), IBA (10 μM) + Kin (0.1 μM) (c), IAA (10 μM) (b, e) or under HF (Insets in a, d, f, g). a Col-0 TCLs showing a poor callus formation and a lot of elongated ARs with hairs. b Col-0 TCLs with elongated hairy ARs, and high callus formation. c Col-0 TCLs with ARPs (arrow) and no elongated AR, and callus. d ech2ibr10 TCLs with a poor number of highly elongated ARs, and a very reduced callus formation. e ech2ibr10 TCLs with callus and elongated ARs. fg wei2wei7 (f) and lax3aux1 (g) TCLs with a very few number of ARs which were not elongated. Insets in a, d, f, and g show the absence of AR formation in the HF-treated control explants. Bars = 1 mm (a-c, e-g, and insets in a, d, f, g), 2 mm (d)
Fig. 3
Fig. 3
IAA-driven DR5::GUS expression in IBA-cultured TCLs, and ASA1 expression under IAA, IBA, and MeJA + IBA. ad Expression in TCLs treated with IBA (10 μM) for 8 (a-b) and 15 days (c-d). a-b Beginning of the signal in early meristemoids (a) and in ARPs (b). c-d DR5-signal in the quiescent centre and some initial and cap cells in the apex of elongating (c) and mature (d) ARs. et ASA1::GUS expression. eg Expression in TCLs observed under the stereomicroscope at day 15 under IAA (10 μM) (e), IBA (10 μM) (f), or MeJA (0.01 μM) + IBA (10 μM) (g), showing differences in signal intensity and localization among the treatments at explant level, but not in the AR apex (Insets). hl Histological analysis of the expression at 8 (h-i) and 15 (j-l) day in IAA-treated TCLs. hi Expression in the endodermis derivatives (h) and in de novo formed xylary cells (arrow), and in the apex of the developing ARPs (i). jk Widespread expression in the ARPs entrapped in the callus (j), and in the apices of the frequently fused ARs (k). l Faint expression in the meristematic cells of the xylogenic nodules. mp Histological analysis of the expression at 8 (m-n) and 15 (o-p) days in IBA-treated TCLs. m–n High expression in the endodermis derivatives (m), and in the apical part of the forming ARPs (n). op Signal in the initial cells of the niche and in the protodermis of the apex of the elongating ARPs (o) and ARs (p), with faint expression in the columella in both cases. qt Histological analysis of the expression at 8 (q-r) and 15 (s-t) days in MeJA + IBA-treated TCLs. qr Strong signal in the endodermis derivatives (q), and in the apical part of the forming ARPs (r). st High signal in the initial cells of the niche and protodermis of the apex of the elongating ARPs (s), and ARs (t), with a lower expression in the columella in both cases. Bars = 20 μm (r, s), 40 μm (a-c, h, i, k-p, q, t), 50 μm (d, j, o), 500 μm (e-g and Insets)
Fig. 4
Fig. 4
Adventitious rooting on TCLs from various genotypes cultured with IBA (10 μM) or IAA (10 μM). a Mean number (±SE) of ARs per IBA- and IAA-cultured TCL of Col-0 and ech2ibr10 at day 15. b Mean number (±SE) of ARs per IBA-cultured TCL of Col-0 and wei2wei7 at day 15. c Mean number (±SE) of ARs per IBA-cultured TCL of Col and lax3aux1 at day 15. a, P < 0.0001 difference with respect to ech2ibr10 within the same treatment; b, P < 0.05 and c, P < 0.0001 difference with respect to IAA within the same genotype; d, P < 0.01 difference with respect to the WT (Col-0 in b, Col in c). Columns with no letter are not significantly different. N = 100
Fig. 5
Fig. 5
Expression pattern of PIN1, LAX3, and AUX1 during AR formation in IBA-cultured TCLs at day15. ae PIN1::GUS expression. a Signal in a wide population of endodermis derivatives and in meristemoids. bc Signal in the basal part of young ARPs (b), all along the developing vasculature (c), and in the central cells of the apex of elongating ARPs (d). e Expression in the vasculature (Inset), and faintly in the apex of mature ARs. fj LAX3::GUS expression. fg Onset of expression in the meristematic cell clusters formed by the endodermis. h Expression at the base of the differentiating ARPs (h). ij Strong LAX3 signal in the procambium (i), and vasculature of the maturing ARs (j). ko AUX1::GUS expression. k–l Uniform signal in the meristematic cell clusters formed by the endodermis (k), and in early primordia (l). m Signal in the cap, protodermis, and developing procambium in the elongating ARPs. n AUX1 expression in the cap, protodermis, and faintly in the niche and procambium (arrow) in a mature AR. o Expression in the vascular parenchyma of the AR primary structure zone. a-e, f-i, k-n, longitudinal sections, j, o, and Inset in e, transections. Bars = 40 μm (a, b, fk, n, o), 50 μm (c-e, l, m, and Inset in e)
Fig. 6
Fig. 6
Detection and quantification of the epifluorescence signal caused by NO in IBA- or IAA-cultured TCLs. ad Presence of the epifluorescence signal (green colour) at 48 h in cells of the deepest layers of TCLs cultured with IBA (10 μM) (a-b), or IAA (10 μM) (c-d). e Numerous endodermis derivative cells showing the NO green signal in TCLs cultured with IBA for 3 days. f Rare cells with a faint signal in the deepest layers of the explant in the presence of IAA at day 3. g Detail of the numerous layers of the endodermis derivatives showing the green epifluorescence at day 6 (IBA treatment). h Presence of the green signal in the first formed ARPs (day 6, IBA treatment). ij Very faint signal in scattered cells (i), and in thin-layered endodermis derivatives (j) of the explant at day 6 (IAA treatment). TCL longitudinal views. The same images under light microscopy are shown in the Insets. k Mean intensity (±SE) of NO fluorescence (AUs) in TCLs cultured with either IBA (10 μM) or IAA (10 μM) for 48 h and 3 days. a,b, P < 0.0001 difference with IAA within the same culture time. c, P < 0.001 difference with the other culture time within the same treatment. Columns with no letter are not significantly different. N = 200. Bars = 50 μm (b, c, e, gj and Insets in b, f, g, i), 70 μm (a, d, f, and Insets in c, e, h, j), 100 μm (Insets in a and d)
Fig. 7
Fig. 7
Model explaining the promotion by exogenous IBA (10 μM) of AR formation in arabidopsis TCLs. Nitric oxide (NO) formed during the exogenous IBA-to-IAA conversion by ECH2/IBR10 induces the synthesis of JA, which, in turn, induces ASA1/ASB1 activity. The IAA, coming from IBA conversion and biosynthesis by ASA1/ASB1, is transported into the target cells of the rhizogenic process by the efflux carrier PIN1 and the influx carriers AUX1 and LAX3. NO might also positively affect PIN1 and AUX1, enhancing the endogenous IAA transport required for adventitious rooting. (See the text for further explanations)

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