Gibberellins accumulate in the elongating endodermal
cells of Arabidopsis root
Eilon Shania,1, Roy Weinstainb,1, Yi Zhanga, Cristina Castillejoa, Eirini Kaiserlic, Joanne Choryc,d,
Roger Y. Tsienb,e,f, and Mark Estellea,f,2
aSection of Cell and Developmental Biology, Departments ofbPharmacology andeChemistry and Biochemistry, andfHoward Hughes Medical Institute,
University of California at San Diego, La Jolla, CA 92093; andcPlant Biology Laboratory, anddHoward Hughes Medical Institute, The Salk Institute for
Biological Studies, La Jolla, CA 92037
Contributed by Mark Estelle, January 10, 2013 (sent for review November 12, 2012)
Plant hormones are small-molecule signaling compounds that are
collectively involved in all aspects of plant growth and develop-
ment. Unlike animals, plants actively regulate the spatial distribu-
tion of several of their hormones. For example, auxin transport
results in the formation of auxin maxima that have a key role in
developmental patterning. However, the spatial distribution of
the other plant hormones, including gibberellic acid (GA), is largely
unknown. To address this, we generated two bioactive fluorescent
GA compounds and studied their distribution in Arabidopsis thali-
ana roots. The labeled GAs specifically accumulated in the endo-
dermal cells of the root elongation zone. Pharmacological studies,
along with examination of mutants affected in endodermal spec-
ification, indicate that GA accumulation is an active and highly
regulated process. Our results strongly suggest the presence of
an active GA transport mechanism that would represent an addi-
tional level of GA regulation.
root development|ethylene|root growth|fluorescent labeling|
mone response pathways at multiple levels including hormone
biosynthesis, metabolism, perception, and signaling. In the case
of auxin, elegant studies have shown that the regulation of auxin
distribution through the action of specific transporters also has
a central role in plant development (2, 3). The recent isolation of
transporters for other hormones (4–6) suggests that the spatial
distribution of these compounds may also be regulated.
Gibberellins (GAs) are a class of tetracyclic diterpenoid hor-
mones that regulate many developmental processes such as seed
germination, root and shoot elongation, flowering and fruit pat-
terning (7–9). Over the years, more than 130 GAs have been
identified, of which only a few, such as GA1, GA3, and GA4, are
bioactive (8). Much progress has been made in understanding how
plants control GA response through regulation of biosynthesis,
metabolism, and signaling (10–14). Although experiments with
radiolabeled GAs as well as grafting studies have established that
GAs move through the plant (15–18), little is known about either
the mechanisms of transport or the distribution of GA.
To address these questions, we generated fluorescently tagged
GA. The labeled GAs retained much of their bioactivity and thus
were used as fluorescent GA surrogates to study the distribution
of GA in the Arabidopsis root system.
daptive growth of plants is regulated by small-molecule
regulators called plant hormones (1). Plants regulate hor-
Fluorescently Labeled GAs Are Bioactive. Four derivatives of fluo-
rescein (Fl)-labeled GA3were synthesized (SI Appendix, Figs.
S1–S4), varying primarily in the length of the linker between Fl
and GA3(Fig. 1A and SI Appendix, Fig. S5A). Conjugation to
GA3through amide formation on C6 was based on previous
reports demonstrating its stability in vitro and in vivo (19). The
four molecules were compared for their GA bioactivity. GAs are
essential for seed germination in many plants including Arabi-
dopsis (20, 21). The GA biosynthesis mutant ga1 germinates
poorly, whereas exogenous application of GA3 fully restores
germination levels (22, 23). Whereas molecules 1 and 4 had a very
small effect on ga1 germination (4% and 8%, respectively),
treatment with molecules 2 and 3 resulted in 33% and 53%
germination. Application of Fl to ga1 plants had no effect on
germination (Fig. 1B and SI Appendix, Fig. S5B). Similarly, for
wild-type (WT) plants treated with the GA-biosynthesis inhibitor
paclobutrazol (Paclo), molecules 2 and 3 showed the highest rate
of germination (38% and 27%, respectively), whereas 1 and 4
had a smaller effect (13% and 16%, respectively) and Fl had no
effect at all (SI Appendix, Fig. S5C). GAs are also key regulators
of hypocotyl and root elongation (24, 25). In 4-d-old WT seed-
lings treated with Paclo, molecules 1, 4, and Fl had little effect on
Paclo-treated hypocotyls whereas 2 and 3 partially restored
elongation (73% and 69%, respectively) (Fig. 1 C and D and SI
Appendix, Fig. S5 D and E). Strikingly, in WT plants treated with
Paclo, application of molecules 2 and 3 fully restored root
elongation, compared with GA3, whereas 1 and 4 had only
a modest effect (Fig. 1E and SI Appendix, Fig. S5F). In this assay,
the half-maximal effective concentration (EC50) of the best
performing derivative molecule 3 (termed “GA3-Fl” from here
on) was 130% that of GA3(1.13 ± 0.02 μM and 0.87 ± 0.11 μM,
respectively) (Fig. 1F). Together, these results demonstrate that
GA3can be labeled with fluorescein and retain biological activ-
ity. Structurally, there is a positive correlation between linker
length and bioactivity. Reintroduction of the carboxylic acid in
close proximity to its original location on GA’s C6 (molecule 4)
did not have a positive effect on bioactivity. To further evaluate
tagged GA function across species, we tested GA3-Fl bioactivity
in Solanum lycopersicum (tomato) compound leaf development.
Tomato plants treated with GA3-Fl presented simpler leaves
with smooth margins, mimicking GA’s effect on tomato leaf
shape (SI Appendix, Fig. S6).
The minor structural differences between GAs prompted us
to test whether the strategy used for GA3labeling is also ef-
fective for other bioactive GAs. Thus, GA4was labeled with
fluorescein similarly to GA3-Fl (Fig. 1G) and tested for its ac-
tivity. The fluorescent conjugate (GA4-Fl) had comparable ac-
tivity to GA4with respect to germination (Fig. 1H) and root and
hypocotyl elongation (SI Appendix, Fig. S7). This suggests that
the GA-labeling strategy described here could be applied to other
GA derivatives. GA3-Fl and GA4-Fl have spectroscopic prop-
erties characteristic of fluorescein (λex= 496 nm, λem= 523 nm,
Φf= 0.81 and λex= 495 nm, λem= 525 nm, Φf= 0.75, respec-
tively) (SI Appendix, Fig. S8), making them suitable for detection
by fluorescence imaging.
Author contributions: E.S., R.W., Y.Z., R.Y.T., and M.E. designed research; E.S., R.W., and
Y.Z. performed research; C.C., E.K., and J.C. contributed new reagents/analytic tools; E.S.,
R.W., Y.Z., R.Y.T., and M.E. analyzed data; and E.S., R.W., R.Y.T., and M.E. wrote
The authors declare no conflict of interest.
See Commentary on page 4443.
1E.S. and R.W. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| March 19, 2013
| vol. 110
| no. 12 www.pnas.org/cgi/doi/10.1073/pnas.1300436110
seedlings were transferred at day 4, and root growth was measured after
a further 5 d. For hypocotyl assays, seedlings were transferred at day 4 and
measured 5 d later. Ten-day-old tomato seedlings were treated three times
a week for 2 wk (shoot apical meristem and small leaves) with liquid solution
of 10 μM of the indicated compounds. The mature third leaf was imaged.
Seed germination and root and hypocotyl growth were all imaged and
measured using a Nikon SMZ1500 dissecting scope and ImageJ software
Complete methods are described in SI Appendix.
ACKNOWLEDGMENTS. We thank Larry Gross (University of California at San
Diego) for mass spectroscopy; Joseph Ecker (The Salk Institute for Biological
Studies) for sharing ein2-5 and ctr1-1 mutants; the Arabidopsis Biological
Research Center (ABRC) (The Ohio State University), which provided Arabi-
dopsis pRGA:GFP-RGA, shr-2, and scr-3 lines; and Tai-ping Sun for the pull-
down pGEX-GID1b (GST-GID1b) plasmid and the yeast two-hybrid DB-GID1a/
AD-RGA plasmids. This work was funded by Vaadia–BARD Postdoctoral Fellow-
ship FI-431-10 (to E.S.); a Machiah Foundation/Jewish Community Federation
Fellowship (E.S.); R25T CRIN Training Grant 5R25CA153915-03 (to R.W.); The
Marc and Eva Stern Foundation (E.K.); Human Frontier Science Program (HFSP)
Fellowship LT000159/2009-L (to E.K.); National Science Foundation Grant IOS10-
45256 (to J.C.); National Institutes of Health Grants 1R01 GM094428 (to J.C.),
NS27177 (to R.Y.T.), and 1R01 GM43644 (to M.E.); Department of Energy
Grant DE-FG02-11ER16007 (to M.E.); the Howard Hughes Medical Institute
(J.C., R.Y.T., and M.E.); and the Gordon and Betty Moore Foundation (M.E.).
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