Enhancement of Indole-3-Acetic Acid Photodegradation by Vitamin B6

Abstract

Dear Editor, The plant hormone indole-3-acetic acid (IAA) has long been used in plant culture media for practical applications and scientific inquiries. The use of IAA is complicated by the fact that IAA is a photo-labile compound. In Murashige and Skoog (MS) plant media (Murashige and Skoog, 1962Murashige T. Skoog F A revised medium for rapid growth and bioassays with tobacco tissue cultures.Physiol. Plant. 1962; 15: 473-497Crossref Scopus (53695) Google Scholar), the concentrations of salts and mineral nutrients are known to hasten the photodegradation of IAA under white light (Dunlap and Robacker, 1988Dunlap J.R. Robacker K.M Nutrient salts promote light-induced degradation of indole-3-acetic acid in tissue culture media.Plant Physiol. 1988; 88: 379-382Crossref PubMed Google Scholar). This degradation can be virtually eliminated by the use of a yellow-colored light filter that removes UV, violet, and some of the blue wavelengths from the incident light (Stasinopoulos and Hangarter, 1990Stasinopoulos T.C. Hangarter R.P Preventing photochemistry in culture media by long-pass light filters alters growth of cultured tissues.Plant Physiol. 1990; 93: 1365-1369Crossref PubMed Scopus (116) Google Scholar). However, the use of yellow light clearly affects the quality of light that the plants under study receive. In addition to applications in plants, IAA has been used in human health applications. The light-induced breakdown product of IAA has cytotoxic properties that make it suitable for the treatment of certain ailments (Folkes and Wardman, 2003Folkes L.K. Wardman P Enhancing the efficacy of photodymanic cancer therapy by radicals from plant auxin (indole-3-acetic acid).Cancer Res. 2003; 63: 776-779PubMed Google Scholar). Photosensitizing dyes are typically employed to aid in the production of this photoproduct (Na et al., 2011Na J.-I. Kim S.-Y. Kim J.-H. Youn S.-W. Huh C.-H. Park K.-C Indole-3-acetic acid: a potential new photosensitizer for photodynamic therapy of acne vulgaris.Laser Surg. Med. 2011; 43: 200-205Crossref PubMed Scopus (34) Google Scholar). We report here that the presence of pyridoxine (PN) in MS media enhances the rate of IAA photodegradation. Although it is known that the degradation rate of IAA is affected by the salt and mineral nutrient composition of MS media, it has not to our knowledge been previously reported that any of the B-vitamins, which are often added to plant culture media, have an effect on the IAA degradation rate. Plants directly produce pyridoxal-5′-phosphate (PLP), the enzymatically active form of vitamin B6 (Titiz et al., 2006Titiz O. Tambasco-Studart M. Warzych E. Apel K. Amrhein N. Laloi C. Fitzpatrick T.B PDX1 is essential for vitamin B6 biosynthesis, development and stress tolerance in Arabidopsis.Plant J. 2006; 48: 933-946Crossref PubMed Scopus (125) Google Scholar), but they also contain PN in similar concentrations as PLP (González et al., 2007González E. Danehower D. Daub M.E Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5’-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway.Plant Physiol. 2007; 145: 985-996Crossref PubMed Scopus (64) Google Scholar). PN is the vitamin B6 vitamer used in plant media, because PLP is highly unstable in light and is not thought to be transportable into plant cells. Vitamin B6 has also been shown to be important for root growth (Chen and Xiong, 2005Chen H. Xiong L Pyridoxine is required for post-embryonic root development and tolerance to osmotic and oxidative stresses.Plant J. 2005; 44: 396-408Crossref PubMed Scopus (139) Google Scholar). The production of IAA depends in part upon tryptophan aminotransferase, an enzyme that utilizes PLP as a cofactor (Won et al., 2011Won C. Shen X. Mashiguchi K. Zheng Z. Dai X. Cheng Y. Kasahara H. Kamiya Y. Chory J. Zhao Y Conversion of tryptophan to indole-3-acetic acid by TRYPTOPHAN AMINOTRANSFERASES OF ARABIDOPSIS and YUCCAs in Arabidopsis.Proc. Natl Acad. Sci. U S A. 2011; 108: 18518-18523Crossref PubMed Scopus (437) Google Scholar). While investigating the combined effects of IAA and vitamin B6 on growth and development of the root uv-b sensitive1 (rus1) mutant (Leasure et al., 2011Leasure C.D. Tong H.Y. Hou X.W. Shelton A. Minton M. Esquerra R. Roje S. Hellmann H. He Z.H root ub-b sensitive mutants are suppressed by specific mutations in ASPARTATE AMINOTRANSFERASE2 and by exogenous vitamin B6.Mol. Plant. 2011; 4: 759-770Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), we observed that PN strikingly diminished the root growth-inhibiting effects of IAA on wild-type Columbia ecotype (WT) seedlings. IAA in high external concentrations (~ 1 µM) causes severe short roots in WT seedlings. We observed that this effect was diminished when PN was added to the media in high concentrations. Our experiments suggest that this phenomenon is due to an increase in the rate of photodegradation of IAA when PN is present in the media. PN increased the rate of IAA degradation under typical growth chamber light conditions (see Supplemental Figure 1 for our precise light intensity measurements), in a dosage-dependent manner. WT seedlings were grown vertically for 4 d in a standard growth chamber (16L:8D day cycles) on MS media (1 × MS salts; 0.3% sucrose; 1% agar; pH 5.85) plates with differing combinations of IAA (0, 1, or 10 µM) and PN (0 or 25 µg ml–1). Plants were grown under standard white light conditions (i.e. no filter) or under a yellow filter (acrylic, Yellow 2208), which blocks lower-wavelength light (Supplemental Figure 1). In the absence of IAA, the light and PN conditions had little to no effect on root growth (Figure 1A and 1B). Under yellow light conditions, 1 and 10 µM IAA essentially eliminated all root growth, regardless of PN presence or absence in the media (Figure 1A and 1B). Under white light conditions, roots grown with 1 or 10 µM IAA (without PN) grew roughly 40% or 20% as long as roots grown without IAA, respectively (Figure 1A and 1B). This increased root growth is due to the partial photodegradation of IAA in the growth media. Under the same white light conditions, the addition of 25 µg ml–1 PN to the media significantly enhanced root growth on both concentrations of IAA (Figure 1A and 1B). The effect of PN on root growth in the presence of IAA is not due to the small changes in pH caused by the addition of PN (which is provided as pyridoxine-HCl), because the addition of equimolar and double-molar amounts HCl in place of PN did not affect root growth (Supplemental Figure 2). The addition of the B-vitamin Thiamine did not affect root growth on IAA (Supplemental Figure 2). The addition of the B-vitamin nicotinic acid did show a slight increase in root growth on IAA, albeit not anywhere near as dramatic as PN (Supplemental Figure 2). This result is intriguing because nicotinic acid and PN both contain a C5:N1 heterocyclic ring structure. Additionally, the root growth-inhibiting effects of the synthetic and photo-stabile auxin 2,4-dichlorophenoxy acetic acid (2,4-D) were not diminished by addition of PN (Supplemental Figure 3). In our next experiment, we created MS plates with 0 or 1 µM IAA in combination with increasing quantities of PN (0, 1, 5, 10, or 25 µg ml–1) (1 µg ml–1 is a typical concentration for PN when used in MS growth media). We placed the plates alone (without seeds) in our growth chamber for 2 d. One full set of plates was placed under white light and another under yellow light. After 2 d, WT seeds were planted on all of the plates, and then both sets of plates were placed vertically under only yellow light. As before, the light and PN conditions had little to no effect on root growth in the absence of IAA (Figure 1C and 1D). Root growth was practically abolished on all of the IAA plates that had been left under yellow light, irrespective of PN concentration (Figure 1C and 1D). For the white light-treated plates containing IAA, the presence of PN increased root growth in a dosage-dependent manner (Figure 1C and 1D). These results suggest that PN increased the rate of IAA degradation during the 2 d of white light treatment that the plates received prior to seeds being placed on them. Therefore, the effect of PN on the degradation rate of IAA appears to occur in the MS media under white light conditions, rather than in the plants themselves. We next analyzed the degradation of IAA in MS media spectrophotometrically. We prepared 200 µM IAA (IAA alone), 200 µM PN (PN alone), and 200 µM IAA with 200 µM PN (IAA+PN) in liquid MS media without agar. As PN itself photodegrades in MS media under white light, we also mixed 200 µM IAA with 200 µM pre-degraded PN (degPN). DegPN was created by placing PN in liquid MS media under white light in our growth chamber for 7 d (Supplemental Figure 4). The result of this experiment was that the daily spectra readings for ‘IAA alone’ suggested that IAA was almost completely degraded after 4 d under white light conditions (Figure 1E). After only 2 d of exposure, the spectra of IAA from ‘IAA+PN’ or ‘IAA+degPN’ appeared similar to the spectrum of ‘IAA alone’ after 4 d (Figure 1E). No IAA degradation was observed under yellow light for any combination (Supplemental Figures 5 and 6). This experiment confirms the root growth experiments, and further suggests that the presence of PN (or even degPN) enhances the rate at which IAA photodegrades under white light. We finally tested to see whether the enhanced degradation of IAA is caused by a light-independent chemical reaction between the degPN and IAA. We mixed degPN and IAA in liquid MS, and left the mixture under yellow light or complete darkness for 4 d. After 4 d, neither the yellow light nor dark conditions showed any degraded IAA (Supplemental Figure 6). From this, we conclude that the enhanced rate of degradation of IAA is not caused by a light-independent reaction with degPN. Therefore, all of our data suggest that IAA degradation is enhanced by PN presence in the media in a manner that remains light-dependent. IAA has also been used as a photodynamic therapy in humans for the treatment of acne vulgaris and certain tumors (Folkes and Wardman, 2003Folkes L.K. Wardman P Enhancing the efficacy of photodymanic cancer therapy by radicals from plant auxin (indole-3-acetic acid).Cancer Res. 2003; 63: 776-779PubMed Google Scholar; Na et al., 2011Na J.-I. Kim S.-Y. Kim J.-H. Youn S.-W. Huh C.-H. Park K.-C Indole-3-acetic acid: a potential new photosensitizer for photodynamic therapy of acne vulgaris.Laser Surg. Med. 2011; 43: 200-205Crossref PubMed Scopus (34) Google Scholar). This treatment involves photo-oxidation of the relatively non-toxic IAA, which produces a compound with some degree of cytotoxicity. The use of photosensitizing dyes is typically employed in this process (Brennan et al., 2000Brennan T.M. Lee E. Battaglia P.R Participation of the photosensitizer alpha-terthienyl in the peroxidase-catalyzed oxidation of indole-3-acetic acid.Photochem. Photobiol. 2000; 71: 355-360Crossref PubMed Scopus (4) Google Scholar). Our studies suggested that PN is an effective photosensitizer for IAA, and may potentially have medical applications. PN is generally considered safe, is naturally occurring in humans, and is widely used in human cosmetics and consumables. We conclude that the rate of IAA photodegradation is enhanced when PN is added to MS growth media (Figure 1). This effect was most dramatic with higher concentrations of PN, but even the standard 1-µg ml–1 concentration of PN had some impact on root growth in the presence of IAA under white light conditions (Figure 1C and 1D). To our knowledge, this chemical interaction between IAA and PN has not been reported before. The fact that PN itself degrades over time in MS media should be of interest to researchers studying mutants with defects in vitamin B6 metabolism. Mutations in the RUS1 and ROOT UV-B SENSITIVE2/WEAK AUXIN RESPONSE (RUS2/WXR) genes cause severely stunted root growth, appear to cause defects in auxin transport, and are largely suppressed by the addition of PN to the growth media (Ge et al., 2010Ge L. Peer W. Robert S. Swarup R. Ye S. Prigge M. Cohen J.D. Friml J. Murphy A. Tang D. et al.Arabidopsis ROOT UVB SENSITIVE2/WEAK AUXIN RESPONSE1 is required for polar auxin transport.Plant Cell. 2010; 22: 1749-1761Crossref PubMed Scopus (35) Google Scholar; Leasure et al., 2011Leasure C.D. Tong H.Y. Hou X.W. Shelton A. Minton M. Esquerra R. Roje S. Hellmann H. He Z.H root ub-b sensitive mutants are suppressed by specific mutations in ASPARTATE AMINOTRANSFERASE2 and by exogenous vitamin B6.Mol. Plant. 2011; 4: 759-770Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Tryptophan aminotransferase enzymes, which utilize PLP as a cofactor, are involved in the production of IAA in plants. Our report of a chemical reaction between IAA and PN, at least in MS growth media, adds more information about the link between these two chemicals. The exact relationship between vitamin B6 and IAA, and whether or not this involves photochemistry in planta, remains to be determined. Supplementary Data are available at Molecular Plant Online.