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Molecular Plant Volume 6 Number 6 Pages 19921995 November 2013

LETTER TO THE EDITOR

Enhancement of Indole-3-Acetic Acid


Photodegradation by Vitamin B6
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, 1962), the concentrations of salts and mineral nutrients are known to hasten
the photodegradation of IAA under white light (Dunlap and
Robacker, 1988). 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, 1990). 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, 2003). Photosensitizing dyes
are typically employed to aid in the production of this photoproduct (Na etal., 2011).
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., 2006), but they
also contain PN in similar concentrations as PLP (Gonzlez
etal., 2007). 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,
2005). The production of IAA depends in part upon tryptophan aminotransferase, an enzyme that utilizes PLP as a
cofactor (Won etal., 2011).
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., 2011), 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 (~ 1M) 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 Figure1 for our precise light intensity measurements), in a dosage-dependent manner.
WT seedlings were grown vertically for 4d 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 10M) and PN (0 or 25gml1).
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 Figure1). In the
absence of IAA, the light and PN conditions had little to no
effect on root growth (Figure1A and 1B). Under yellow light
conditions, 1 and 10 M IAA essentially eliminated all root
growth, regardless of PN presence or absence in the media
(Figure1A and 1B). Under white light conditions, roots grown
with 1 or 10M IAA (without PN) grew roughly 40% or 20%
as long as roots grown without IAA, respectively (Figure1A
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 25gml1 PN to the
media significantly enhanced root growth on both concentrations of IAA (Figure1A 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 Figure2). 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 Figure3).

The Author 2013. Published by the Molecular Plant Shanghai Editorial


Office in association with Oxford University Press on behalf of CSPB and
IPPE, SIBS,CAS.
doi:10.1093/mp/sst089, Advance Access publication 30 May 2013
Received 14 March 2013; accepted 20 May 2013

Letter to the Editor

1993

Figure1. IAA Photodegradation Is Enhanced by PN.


(A) Representative 4-day-old WT seedlings grown on 0, 1, or 10M IAA, with or without PN (25gml1), and under white light (WL) or yellow
light (YL) conditions. Bar=5mm.
(B) Graph of mean root length of 4-day-old WT seedlings grown on 0, 1, or 10M IAA, with or without PN (25gml1), and under white light (WL)
or yellow light (YL) conditions. Error bars=standard error. n=10 for all conditions, except for 10M IAA/WL/0 PN n=9.
(C) Representative 4-day-old WT seedlings grown under YL on 1M IAA, with varying concentrations of PN (025gml1). Plates were treated for
2d prior to planting seeds with white light (WL) or yellow light (YL). Bar=5mm.
(D) Graph of mean root length of 4-day-old WT seedlings grown under YL on 1M IAA, with varying concentrations of PN (025gml1). Plates
were treated for 2d prior to planting seeds with white light (WL) or yellow light (YL). Error bars=standard error. n=810.
(E) Spectra of 200M IAA in liquid MS media after exposure to 0, 24, 48, or 96h of white light. Blue line=IAA alone. Red line=IAA from the
mixture of 200M IAA + 200M PN (obtained by subtracting out the absorbance due to 200M PN). Green line=IAA from the mixture of 200M
IAA + 200M degPN (obtained by subtracting out the absorbance due to 200M degPN).

1994

Letter to the Editor

In our next experiment, we created MS plates with 0 or


1M IAA in combination with increasing quantities of PN (0,
1, 5, 10, or 25gml1) (1gml1 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 2d, 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 (Figure1C and 1D). Root growth was practically abolished on all of the IAA plates that had been left under yellow
light, irrespective of PN concentration (Figure1C and 1D). For
the white light-treated plates containing IAA, the presence
of PN increased root growth in a dosage-dependent manner
(Figure1C 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 M IAA (IAA
alone), 200M PN (PN alone), and 200M IAA with 200M
PN (IAA+PN) in liquid MS media without agar. As PN itself
photodegrades in MS media under white light, we also mixed
200 M IAA with 200 M pre-degraded PN (degPN). DegPN
was created by placing PN in liquid MS media under white
light in our growth chamber for 7d (Supplemental Figure4).
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 whitelight.
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 4d. After 4d, neither the yellow light nor
dark conditions showed any degraded IAA (Supplemental
Figure6). 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, 2003; Na etal., 2011). 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 etal., 2000). 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 (Figure1).
This effect was most dramatic with higher concentrations of
PN, but even the standard 1-gml1 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., 2010; Leasure et al., 2011).
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.

SUPPLEMENTARYDATA
Supplementary Data are available at Molecular Plant Online.

FUNDING
This work was supported by the National Institute of General
Medical Sciences (NIH Award Number SC1GM095462). No
conflict of interest declared.

Colin D.Leasure1, Yi-PeiChen, and Zheng-HuiHe


Department of Biology, San Francisco State University, San Francisco,
CA 94132, USA To whom correspondence should be addressed. E-mail
cleasure@sfsu.edu, tel. 415-338-6487.

References
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the photosensitizer alpha-terthienyl in the peroxidase-catalyzed oxidation of indole-3-acetic acid. Photochem. Photobiol.
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Letter to the Editor

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