Microbial Metabolite; Endogenous Metabolite

Auxin is a crucial phytohormone involved in multiple plant developmental processes. Spatiotemporal regulation of auxin levels is necessary to achieve development of organs in the proper place and at the proper time. These levels can be regulated by conversion of auxin [indole 3-acetic acid (IAA)] from its conjugated forms and its precursors. Indole-3-butyric acid (IBA) is an auxin precursor that is converted to IAA in a peroxisomal β-oxidation process. In Arabidopsis, altered IBA-to-IAA conversion leads to multiple plant defects, indicating that IBA contributes to auxin homeostasis in critical ways. Like IAA, IBA and its conjugates can be transported in plants, yet many IBA carriers still need to be identified. In this review, we discuss IBA transporters identified in Arabidopsis thus far, including the pleiotropic drug resistance (PDR) members of the G subfamily of ATP-binding cassette transporter (ABCG) family, the TRANSPORTER OF IBA1 (TOB1) member of the major facilitator superfamily (MFS) family and hypothesize other potential IBA carriers involved in plant development. [1]

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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 Indole-3-butyric acid/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 Indole-3-butyric acid/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.[2]

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Controlled plant growth requires regulation through a variety of signaling molecules, including steroids, peptides, radicals of oxygen and nitrogen, as well as the 'classical' phytohormone groups. Auxin is critical for the control of plant growth and also orchestrates many developmental processes, such as the formation of new roots. It modulates root architecture both slowly, through actions at the transcriptional level and, more rapidly, by mechanisms targeting primarily plasma membrane sensory systems and intracellular signaling pathways. The latter reactions use several second messengers, including Ca(2+) , nitric oxide (NO) and reactive oxygen species (ROS). Here, we investigated the different roles of two auxins, the major auxin indole-3-acetic acid (IAA) and another endogenous auxin indole-3-butyric acid (IBA), in the lateral root formation process of Arabidopsis and maize. This was mainly analyzed by different types of fluorescence microscopy and inhibitors of NO production. This study revealed that peroxisomal IBA to IAA conversion is followed by peroxisomal NO, which is important for IBA-induced lateral root formation. We conclude that peroxisomal NO emerges as a new player in auxin-induced root organogenesis. In particular, the spatially and temporally coordinated release of NO and IAA from peroxisomes is behind the strong promotion of lateral root formation via IBA.[3]

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In vitro activity: The total yield of chicks during the good laying seasons of the year is about 80 to 85%, based on a high percent of hatchable eggs of about 80 to 90%. The total yield of chicks during the poor laying seasons of the year is about 50 to 55%, based on a high percent of hatchable eggs of about 60 to 65%. In well run commercial hatcheries, it is considered excellent that an average yield for any one year of the total number of incubated eggs is about 75%. Cell Assay: To eggs that was naturally fertilized and showed no sign of a living embryo prior, treatment with IBA in optimum amounts facilitated the live embryo formation in certain of the eggs. It further increased the chick yield of a given batch of eggs. In the life-giving cells of the blastoderm of the egg, IBA in optimum amounts stimulated the life growing activity of the cells and resulted in the formation of a live embryo, though the formation failed to show before treatment under the most careful candling.

To the live animal embryo of a fertilized avian egg, when Indole-3-butyric acid/IBA is made available, it stimulated the biological life-growing processes during incubation. In all cases, after the treatment of IBA, hatched chicks were healthy, viable and vigorous. They tended to be more resistant to disease than common chicks hatched from untreated eggs

TCL culture [2]
Superficial TCLs, about 0.5 × 8 mm, composed by six cell layers including the epidermis, were excised from the internodes of the inflorescence stem. The TCLs were cultured, epidermal side up, under continuous darkness, at 22 ± 2 °C, up to day 15 on a medium consisting of MS salts supplemented with 0.55 mM myo-inositol, 0.1 μM thiamine-HCL, 1% (w/v) sucrose, 0.8% agar (w/v) (pH 5.7) (HF medium). Col-0 TCLs were cultured on this medium with the addition of 10 μM IBA/Indole-3-butyric acid, 10 μM IBA plus 0.1 μM Kin, 0.1 μM Kin, 10 μM IAA, 10 μM IBA plus 0.01 μM MeJA, and under HF as experimental control. TCLs from the ech2ibr10, wei2-1wei7-1, and lax3aux1-21 mutants and their WT were cultured with 10 μM IBA, 10 μM IAA or under HF. ASA1::GUS TCLs were cultured with either 10 μM IBA, or 10 μM IAA or IBA plus 0.01 μM MeJA. DR5::GUS, PIN1::GUS, LAX3::GUS, AUX1::GUS TCLs, and TCLs from Col and Col-gl1 ecotypes, were cultured with 10 μM IBA. One hundred explants per genotype and treatment were used per replicate. The pH was adjusted to 5.7 with 1 M NaOH before autoclaving. For macroscopic analyses, the explants of the WT and mutants were examined under a LEICA MZ8 stereomicroscope at culture end, and the AR response evaluated as the percentage of explants either remaining at the initial stage at culture end or forming macroscopic callus and ARs, and as mean number of ARs (±SE) per rooting explant.

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Histochemical analysis of GUS activity [2]
TCLs of DR5::GUS, PIN1::GUS, LAX3::GUS, AUX1::GUS, ASA1::GUS lines were harvested at day 8 and day 15 of culture, and processed with the GUS staining as described by Willemsen et al, with minor modifications, as reported by Veloccia et al. After infiltration for 15 min in a vacuum belljar, the samples were incubated at 37 °C in the dark either for 30 min (DR5::GUS and LAX3::GUS), or 45 min (AUX1::GUS, ASA1::GUS), or 2.5 h (PIN1::GUS). After GUS assay, the samples were fixed in 70% (v/v) ethanol, dehydrated by a graded ethanol series, embedded in Technovit 7100, longitudinally sectioned at 12 μm with a Microm HM 350 SV microtome, and observed under light microscopy.

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Hormone quantification [2]
TCLs of Col-0 were collected at time 0 (i.e., soon after the excision) and conserved to −80 °C until the analyses. The extraction of IAA and IBA/Indole-3-butyric acid was performed using aliquots of 50 mg of TCLs according to Veloccia et al. Quantitative determinations of IAA and IBA were carried out by Rapid Resolution-Reversed Phase-HPLC (RR-RP-HPLC) separation followed by MS/MS detection with a triple quadrupole (QqQ) mass-spectrometer with an ESI-interface. Pure standards, internal standards, and the quantification of the two auxins were according to Veloccia et al.

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Nitric oxide detection [2]
Intracellular NO content in Col-0 TCLs cultured with either 10 μM IBA or 10 μM IAA was quantified using the cell-permeable diacetate derivative diamino-fluorescein-FM (DAF-FMDA) under epifluorescence microscopy. TCLs were incubated in 20 mM HEPES/NaOH buffer (pH 7.4) supplemented with 5 μM DAF-FMDA for 20 min at 2, 3 and 6 days of culture, after having verified that no significant epifluorescence signal was detectable with the buffer alone (Additional file 1: Figure S1 a-b). After washing three times with the buffer to remove the excess of the fluorescent probe, TCLs were observed using a Leica DMRB microscope equipped with the specific set of filters (EX 450–490, DM 510, LP 515). The images were acquired with a LEICA DC500 digital camera and analysed with the IM1000 image-analysis software. Ten observations in each of 20 TCLs per treatment were randomly carried out, and the intensities of the fluorescence signal (in green colour) were quantified using the ImageJ software and expressed in Arbitrary Units (AUs; from 0 to 255). The values were averaged and normalized to the control ones, i.e., to those measured in TCLs incubated in the buffer without the fluorescent probe.

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Maize grains (Zea mays L.) of wild-type and the lrt1 mutant were soaked for 6 h and germinated on well-moistened rolls of filter paper for 4 d in the dark at room temperature. Seedlings with straight primary roots, 50–70 mm in length (wild-type, lrt1), were selected for auxin treatments. For pharmacological experiments, root apices were submerged in appropriate solutions in the dark at room temperature. For the treatments of roots with auxin, an effective working concentration of 10 μM IAA or IBA/Indole-3-butyric acid was prepared immediately before submerging root apices for 2 h.

Arabidopsis seeds were surface sterilized and placed on half-strength Murashige & Skoog (1962) (MS) culture medium without vitamins and containing 1% sucrose (1.5% for β-oxidation-defective mutants and associated wild-type controls) which was solidified with 0.8% phytagel. Plates with seeds were stored at 4°C for 48 h to break dormancy and then vertically mounted under continuous yellow light for 3–4 d.

Arabidopsis mutants pex5-1 pex7-1, pxa1-1 and pex6 (originally designated B11 by Zolman et al., 2000), with defects in β-oxidation and showing IBA/Indole-3-butyric acid insensitivity (Zolman et al., 2001a; Zolman & Bartel, 2004; Woodward & Bartel, 2005b), and the mutant noa1 with impaired NO production (originally designated Atnos1 by Guo et al., 2003), were used for our experiments.

For microscopy, 3–4-d-old seedlings were transferred to microscopic slides, which were placed in thin chambers made of coverslips. The chambers were filled with half-strength MS medium, but without phytagel, and placed in sterile glass cuvettes containing the medium at a level that reached the open lower end of the chambers. This allowed free exchange of medium to take place between the chambers and the cuvette. Seedlings were grown in a vertical position under continuous yellow light for up to 24 h. During this period, the seedlings stabilized their root growth and generated new root hairs in the liquid medium. In vivo monitoring of peroxisomes was performed with peroxisome targeting signal 1-green fluorescent protein (PTS1-GFP) (see Woodward & Bartel, 2005b).

Non-Human Toxicity Values
LD50 Mouse oral 100 mg/kg
LD50 Mouse ip 100 mg/kg

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8617 rat LD oral >500 mg/kg National Academy of Sciences, National Research Council, Chemical-Biological Coordination Center, Review., 5(7), 1953
8617 mouse LD50 oral 100 mg/kg Agricultural Chemicals, Thomson, W.T., 4 vols., Fresno, CA, Thomson Publications, 1976/77 revision, 3(76), 1976/1977
8617 mouse LD50 intraperitoneal 100 mg/kg Agrochemicals Handbook, with updates, Hartley, D., and H. Kidd, eds., Nottingham, Royal Soc of Chemistry, 1983-86, A231(1983)

[1]. Indole 3-Butyric Acid Metabolism and Transport in Arabidopsis thaliana. Front Plant Sci. 2019 Jul 3;10:851.
[2]. 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. BMC Plant Biol. 2017 Jul 11;17(1):121.
[3]. Indole-3-butyric acid induces lateral root formation via peroxisome-derived indole-3-acetic acid and nitric oxide. New Phytol. 2013 Oct;200(2):473-482.

Indole-3-butyric acid is a indol-3-yl carboxylic acid that is butanoic acid carrying a 1H-indol-3-yl substituent at position 1. It has a role as a plant hormone, a plant metabolite and an auxin. It is functionally related to a butyric acid. It is a conjugate acid of an indole-3-butyrate.
Indole-3-butyric acid has been reported in Zea mays, Cocos nucifera, and other organisms with data available.

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IBA is the main player of adventitious rooting in arabidopsis TCLs, and possibly in many other culture systems and species characterized by very low endogenous auxin contents. IBA acts by conversion into IAA, and by enhancing IAA biosynthesis and transport. The nodal point of its action is the regulation of the endogenous IAA pool. IBA-regulation of IAA homeostasis involves the activity of other compounds downstream to its peroxisomal conversion, NO and jasmonates. The relationship of IBA with NO and jasmonates, and the downstream auxin signalling and perception, needs further investigation. Even if useful for planning experiments to overcome the rooting recalcitrance of species of economic value, the main implication of the findings is to help in understanding the mechanism by which IBA controls the natural process of adventitious rooting,[2]

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A question which still remains to be answered is the role of endogenous IBA. Recent studies have indicated that the IBA to IAA conversion is relevant for undisturbed seedling development by feeding into internal active auxin pools (Strader et al., 2011). One phenotypic aspect of many mutants that cannot utilize IBA is a shortage of lateral roots (e.g. Zolman et al., 2001a,b; Wiszniewski et al., 2008; Strader et al., 2010, 2011). This could be an indicator that IBA to IAA conversion is necessary for LRF. Our present data show that the spatially and temporally coordinated release of NO and IAA is needed for IBA bioactivity. IBA to IAA peroxisomal conversion not only results in tightly regulated IAA synthesis, but also promotes concomitant NO formation. It can be expected that other processes also requiring IBA, and also involving NO, will be shown to rely on peroxisomal IAA–NO generation. Here, we can mention polarized tip growth of root hairs and root adaptation to salt and water stresses (Lombardo et al., 2006; Strader et al., 2010, 2011; Tognetti et al., 2010; Strader & Bartel, 2011). Future work is necessary to elaborate what kind of NO-producing enzyme systems are responsible for the NO formation induced by IBA, and whether NO-mediated signaling is also utilized in other processes in which IBA is active or essential.[3]

C12H13NO2

203.24

203.094

C, 70.92; H, 6.45; N, 6.89; O, 15.74

133-32-4

8617

White to slightly yellow crystals
White to tan powder or crystalline solid

1.3±0.1 g/cm3

426.6±20.0 °C at 760 mmHg

124-125.5 °C(lit.)

211.8±21.8 °C

0.0±1.1 mmHg at 25°C

1.646

2.34

2

2

4

15

230

0

O([H])C(C([H])([H])C([H])([H])C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12)=O

JTEDVYBZBROSJT-UHFFFAOYSA-N

InChI=1S/C12H13NO2/c14-12(15)7-3-4-9-8-13-11-6-2-1-5-10(9)11/h1-2,5-6,8,13H,3-4,7H2,(H,14,15)

4-(1H-indol-3-yl)butanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.30 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)