Supramolecular cation transporters alter root morphology in the Arabidopsis thaliana plant

Article abstract of DOI:10.1016/j.ica.2017.05.019

Bibracchial (two-armed) 4,13-diaza-18-crown-6 lariat ether and tris(macrocycle) hydraphile synthetic amphiphiles alter root morphology in Arabidopsis thaliana plants. The effect on root structure and growth depends both on the hydraphile spacer chain length and lariat ether side chain length as well as the concentration of compound in the growth medium. In some cases a correlation to ion transport activity was apparent, but such a correlation is not always manifested. Surprisingly, planar bilayer conductance (BLM) studies showed that lariat ethers and lariat ether amides both exhibited well controlled membrane activity. Pore formation in soybean asolectin membranes occurred readily and the pores were stable and sustained. Low concentrations of active hydraphiles and lariat ethers altered the primary:lateral root density ratio, generally increasing it. The transporter-mediated alterations in lateral root density were suggestive of the activity of plant auxins such as indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid, which are known to depend on cytosolic potassium ion concentrations. The hypothesis that the compounds interfere with the auxin pathway was tested and discounted by using auxin-resistant A. thaliana mutants. Rather than functioning directly as auxin mimics, ionophores affect the ion gradients producing an auxin-like effect on root development.

Full text of DOI:10.1016/j.ica.2017.05.019

Accepted Manuscript
Research paper
Supramolecular Cation Transporters Alter Root Morphology in the Arabidopsis
Thaliana Plant
Mohit B. Patel, Saeedeh Negin, Ariel Stavri, George W. Gokel
PII:
S0020-1693(17)30334-1
http://dx.doi.org/10.1016/j.ica.2017.05.019
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Please cite this article as: M.B. Patel, S. Negin, A. Stavri, G.W. Gokel, Supramolecular Cation Transporters Alter
Root Morphology in the Arabidopsis Thaliana Plant, Inorganica Chimica Acta (2017), doi: http://dx.doi.org/
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Supramolecular Cation Transporters Alter Root
Morphology in the Arabidopsis Thaliana Plant
‡
¶
†¶
‡
†‡¶
Mohit B. Patel, Saeedeh Negin, Ariel Stavri, and George W. Gokel *
¶
†
Center for Nanoscience, Departments of Chemistry & Biochemistry
‡
and Biology, University of Missouri – St. Louis,
1
University Blvd., St. Louis, MO 63121 U. S. A.
Submitted to Inorganica Chimica Acta
March 3, 2017 Revised May 5, 2017
Version printed: Thu, May 11, 17 at 11:42
*
Corresponding author
G. W. Gokel
Center for Nanoscience
Department of Chemistry & Biochemistry
University of Missouri–St. Louis
St. Louis, MO 63121 USA
Tel. 314/516-5321
FAX 314/516-5342
E-mail: gokelg@umsl.edu
Emails:
gokelg@umsl.edu
mbpyt7@mail.umsl.edu
sn4yd@mail.umsl.edu
astavri@gmail.com
1

Abstract
Bibracchial (two-armed) 4,13-diaza-18-crown-6 lariat ether and tris(macrocycle)
hydraphile synthetic amphiphiles alter root morphology in Arabidopsis thaliana plants.
The effect on root structure and growth depends both on the hydraphile spacer chain
length and lariat ether side chain length as well as the concentration of compound in the
growth medium. In some cases a correlation to ion transport activity was apparent, but
such a correlation is not always manifested. Surprisingly, planar bilayer conductance
(
BLM) studies showed that lariat ethers and lariat ether amides both exhibited well
controlled membrane activity. Pore formation in soybean asolectin membranes occurred
readily and the pores were stable and sustained. Low concentrations of active
hydraphiles and lariat ethers altered the primary:lateral root density ratio, generally
increasing it. The transporter-mediated alterations in lateral root density were
suggestive of the activity of plant auxins such as indole-3-acetic acid and 2,4-
dichlorophenoxyacetic acid, which are known to depend on cytosolic potassium ion
concentrations. The hypothesis that the compounds interfere with the auxin pathway
was tested and discounted by using auxin-resistant A. thaliana mutants. Rather than
functioning directly as auxin mimics, ionophores affect the ion gradients producing an
auxin-like effect on root development.
Graphical Abstract
Keywords:
Arabidopsis thaliana
Auxin
Hydraphile
Ion channel
Ionophore
Lariat ether
Planar bilayer conductance
Plant growth
Pore formation
Root morphology
2

Introduction
Crown ethers have been known and extensively studied for more than four decades. [1]
A spectacular surge in new structures followed Pedersen’s seminal discovery of synthetic
alkali metal binders. [2] [3] [4] Macrocycles having varied sizes and donor groups form
a range of stable and selective complexes with metal and organic cations. [5] Numerous
crown ethers have been reported to transport cations through bulk liquid [6] and bilayer
membranes. [7] A series of dibenzo-18-crown-6 [8] and N,N’-dialkyldiaza-18-crown-6
+
+
ethers or lariat ethers [9] are known to transport Na and K . In addition, the biological
activity of some crown ethers has been known for decades, but such studies have mostly
focused on toxicity. [10] Toxicity studies against various microbes, for example, have
been extensive [11] and are ongoing. There has also been considerable interest in
developing synthetic ion channels of various structural types, including membrane-
spanning and self-assembled variants.[12]
The increase in complexity and sophistication of crown ether structures [13] and their
ability to mimic such natural ionophores as valinomycin, [14] has led to in vivo
applications to mimic biological processes. [15] [16] Several conventional crown ethers
and their derivatives have shown antiproliferative/antitumor properties. [17] The N,N’-
dialkyldiaza-18-crown-6 lariat ether compounds that we have studied show varied
biological activity including toxicity to diverse microbes.[18] The lariat ethers show
toxicity to Gram negative Escherichia coli, Gram positive Bacillus subtilis, and to the
primary eukaryotic yeast Saccharomyces cerevisiae. Although very few complex
organism studies, especially with plants, have been reported, it is known that benzo-15-
+
crown-5, at low concentrations, transports K ions in wheat roots. [19] In other work, it
was found that benzo-18-crown-6 reduced potassium efflux, transport, and uptake in
onion root segments. [20] The leaves of the Asian day lily (Commelina communis) were
studied in the presence of benzo-18-crown-6. [21] Differences were observed in effects
on the upper (abaxial) and lower (adaxial) leaf surfaces that correlated to macrocycle
administration. The authors also noted that the crown had a similar effect on stomata as
observed with the plant auxin phenylacetic acid.
Many of the synthetic channel compounds that we call hydraphiles are biologically
active. [22][23][24][25] Our observation of some similarity in biological effect on
Arabidopsis thaliana between lariat ethers and hydraphile compounds was surprising.
We expected lariat ethers that function as ion carriers to be much less active than
hydraphiles, which are synthetic ion channels (pore-formers). We expanded our
preliminary work on hydraphiles [26] to include lariat ethers that show effects on plant
growth that are remarkably similar to those exhibited by hydraphiles. We have
hypothesized mechanisms for this biological activity either in terms of ion binding and
transport or direct involvement in an auxin (hormone) regulated pathway. We report
here clear planar bilayer conductance evidence for lariat ether pore formation and we
examine possible auxin-like behavior.
Results and Discussion
Higher plants exhibit diverse root architectures [27] and elastic root development.
These properties are important for the extraction of water, macronutrients, and
micronutrients from the environment and for plant anchorage. [28] A. thaliana is the
best characterized plant in terms of genetic, hormonal, and nutritional control of lateral
3

root development. [29] It typically grows with a single primary root from which the
lateral roots extend. Extracellular potassium ion concentration and ion transport have
been reported to affect the development of Arabidopsis roots. [30] We thus initially
hypothesized that lariat ethers and hydraphiles, as ion transporters, might affect growth
and root architecture by altering ion concentrations. This seemed reasonable in light of
our previous findings of the effect of hydraphiles on root structure. [26]
Compounds used in the study. The work described here used two types of synthetic
amphiphiles: hydraphiles [31] and N,N'-disubstituted-4,13-diaza-18-crown-6 two-
armed lariat ethers. [32] [33] The three hydraphiles (previously reported [25]) are
identical to each other except for the spacer chain lengths, which are octylene (C ),
8
tetradecylene (C14), and hexadecylene (C16) respectively in compounds 1-3. Each lariat
ether side chain is attached to a macroring nitrogen atom. In compounds 4-8, the side
chain is an n-alkyl group and in 9-13, the alkyl group is linked via an amide. Two
additional compounds were used in this study. They are the natural and synthetic plant
auxins indoleacetic acid (IAA) 14 and 2,4-dichlorophenoxyacetic acid (2,4-D), 15. The
structures are shown in Figure 1.
O
O
O
O
O
O
O
O
O
O
O
O
CH
2
N
N
(CH
2
)
n
N
N
(CH2)n
N
N
CH2
1
, n = 8; 2, n = 14; 3, n = 16
O
O
O
4, R = C
6
H
13
17
9, R = CO-C
10, R = CO-C H
5
H
11
15
19
5
, R = C H
8
7
R
N
N
R
6, R = C10
H
21
11, R = CO-C
9
H
7
, R = C12H25
12, R = CO-C11H23
O
8, R = C14
H
29
13, R = CO-C13H
27
O
O
OH
O
OH
15
N
H
14
Cl
Cl
Figure 1. Structures of hydraphiles 1-3, lariat ethers of various chain
lengths, without (compounds 4-8) or with (compounds 9-13) amide
residues. Indole-3-acetic acid (14) and 2,4-dichlorophenoxyacetic acid
(
15) are known plant auxins.
We have recently reported an improved preparation of benzyl hydraphiles 1-3. [34] It
was used to prepare the hydraphiles reported here. The synthesis of the lariat ether
amides (9-13) was accomplished by diacylation of the diazacrown with the appropriate
acid chloride. Part of each amide product was reduced using LiAlH
4
or BH •THF,
3
affording the n-alkyl analogs, 4-8. The sequence is shown in Scheme 1 below and details
of the syntheses and products are recorded in the Experimental Section.
4

Scheme 1. Synthesis of lariat ether amides and their reduction to dialkyl
lariat ethers.
Plant growth. A. thaliana Col-0, which is the most common strain of this plant, was
used as the test organism. After sterilizing, the seeds were plated on plant nutrient
media including 0.5% sucrose, (“PNS”) and which contained 0.6% agar. Each
experiment involved 20-30 plants and each experiment was repeated at least three
times (minimum 60-90 plants for each data point). The primary root length for each
plant was measured 11 days after initial incubation and the data obtained are reported
herein below in millimeters. A dissecting microscope was used to assist in counting the
number of lateral roots emerging from the primary root of each plant. We found that
certain hydraphiles and lariat ethers required addition of DMSO to the media for
solubility reasons. It was therefore necessary to confirm that the amount of cosolvent
added was inconsequential because DMSO can affect membrane permeability [35] [36]
and biological activity [37] at higher concentrations. The average primary root length
observed was 42 ± 8 mm when A. thaliana was grown on PNS media. In the presence of
0
.2 volume-% DMSO, the primary root length of A. thaliana was 38 ± 4 mm, which is
within experimental error of PNS alone.
We next conducted experiments to confirm the known effects [38] of the auxins IAA
(
14) and 2,4-D (15) on root development in A. thaliana. The results are recorded in
Table 1. Here, the lateral root density is defined as [(number of lateral roots)/(primary
root length in mm)]. Thus, the first line in Table 1, for PNS media alone, gives a lateral
root density of 6.9/42 = 0.16.
Table 1. Effect of 14 and 15 on A. thaliana root development
Decrease in
primary root
length (%)
A. thaliana (Col-0) Primary root
Number of Lateral root
plant
length (mm)
lateral roots
density
PNS media
42 ± 8
not applicable
6.9 ± 0.2
0.16
5

DMSO (0.2%)
IAA (14) 200 nM
IAA (14) 1 µM
38 ± 6
7.6 ± 2.3
2 ± 1
10
67
85
93
96
6.3 ± 0.1
3.7 ± 1.3
1.9 ± 1.4
3.7 ± 0.3
1.8 ± 0.6
0.16
0.48
0.91
1.19
1.14
2
2
,4-D (15) 200 nM
,4-D (15) 1 µM
3.1 ± 0.1
1.6 ± 0.3
Indoleacetic acid (IAA, 14) showed, as expected for this known auxin, [39] a
concentration dependent effect both on primary root length and lateral root density.
When IAA was administered at a concentration of 200 nM, the primary root length was
7.6 ± 2.3 mm and the lateral root density was 0.48. However, when [IAA] = 1 µM, the
primary root length decreased by 85% and lateral root density increased to 0.91. In the
presence of 2,4-D (15), the primary root lengths at concentrations of 200 nM and 1 µM
were, respectively, 3.1 ± 0.1 mm and 1.6 ± 0.3 mm. The lateral root density at both
concentrations was ~1.1. The synthetic auxin 2,4-D has been reported to alter root
morphology at concentrations as low as 100 nM, [38] so no major change was expected
or observed between 200 nM and 1 µM. These control studies gave results
corresponding to those reported by others.
Effect of hydraphiles on A. thaliana root morphology. We have previously
reported that hydraphiles 1-3, when added to PNS media at a concentration of 20 µM
and 50 µM, affected the root architecture of A. thaliana.[26] After 11 days, the primary
root lengths were diminished with a corresponding increase in lateral root density
(
Supplementary table 1). The change in root architecture by hydraphiles showed an
apparent dependence on the ability of these compounds to transport ions. [40] [41] It is
obvious from the concentrations used that the natural auxin IAA (14) and its synthetic
mimic 2,4-D (15) are substantially more active than the hydraphiles. The amphiphiles
reported here are structurally unrelated to 14 or 15 and were tested because they were
expected to influence ion transport rather than to be auxin mimics.
It seemed possible that the presence of hydraphiles in the growth medium could affect
A. thaliana seed germination. We therefore added varying concentrations of benzyl C16
hydraphile, 3, during germination. The results, shown in the graph of Figure 2, suggest
that germination is indifferent to the presence of 3 in the concentration range 0.5-50
µM. Error bars in the graph have been omitted for clarity.
6

110
100
PNS
9
0
+
+
+
+
+
+
DMSO
0.5 µM
80
3
70
1 µM
5 µM
3
3
60
50
40
10 µM
20 µM
3
30
3
3
20
10
+50 µM
0
0
20
40
60
80
Hours
Figure 2. Germination of A. thaliana in the absence or presence of 0.2
volume-% DMSO or 0.5-50 µM 3. Each point represents the average of
plants grown from 17-25 seeds in each case. PNS is plant growth media
plus sucrose. Error bars have been deleted for clarity.
The fact that hydraphiles 2 and 3 that are known to be effective pore-formers were more
active and more toxic than poor ion-transporter 1, suggested the importance of a pore
formation mechanism. The simpler, but related, lariat ethers [42] are generally known
to be ion carriers, [43] but not pore formers. We therefore explored their activity as a
probe of mechanism and toxic effect.
Effect of lariat ethers on A. thaliana root morphology. The N,N’-disubstituted
4
,13-diaza-18-crown-6 compounds shown in Figure 1 as 4-13 were incorporated into A.
thaliana growth media at the indicated concentrations. Figure 3 shows the effect of n-
alkyl lariats 4 to 8 on the primary root lengths of A. thaliana plants. Each lariat ether
(
4-8) was tested at concentrations of 20 µM (solid line) and 50 µM (dotted line). The
abscissa records the number of CH
2
groups in the side arms of 4 to 8. The ordinate
7

shows the average primary root length in mm.
Figure 3. Effect of lariat ethers (compounds 4-8) at 20 µM (solid line)
and 50 µM (dotted line) on primary root length. Each data point
represents the average of 3 to 4 trials each with 18-23 plants tested per
experiment (total sample 54-92 plants). The error bars represent the
standard error in each experiment.
We did not expect any significant effect of lariats on plant morphology. However, if any
effect was witnessed, we anticipated that it would parallel the ion transport ability
known for these compounds. In fact, the only substantial effects observed at 20 µM
were for 5 (C
presence of 4, 7 (C12), and 8 (C14) were similar to those of PNS control (42 ± 8 mm). At
0 µM, the primary root lengths in the presence of 4, 7, and 8 were 34 ± 2 mm, 38 ± 3
8
) and 6 (C10). At 20 µM concentrations, the primary root lengths in the
5
mm, and 21 ± 2 mm, respectively. The effects of compounds 4-7 at either 20 µM or 50
µM were similar, but not identical. A surprise was that 8 had essentially no effect on
plant growth at 20 µM, but showed a dramatic decrease in primary root length at 50
µM.
The graph of Figure 4 plots the effect of 4-8 on A. thaliana lateral root density as a
function of lariat side chain length. When administered at a concentration of 20 µM, the
lateral root densities for 4, 7, and 8 were 0.16 ± 0.01, 0.15 ± 0.02, and 0.2 ± 0.02,
respectively. At 50 µM, the corresponding values were similar to those observed at 20
µM and little different from those apparent in PNS alone (0.16). As was the case with
primary root length, 5 and 6 showed a significant increase in lateral root density at 20
µM. The variation was similar, but amplified, at 50 µM.
Figure 4. Effect of lariat ethers (compounds 4-8) at 20 µM (solid line)
and 50 µM (dotted line) on the lateral root density of A. thaliana. Each
data point represents the average of 3 to 4 trials each with 18-23 (total 54-
92) plants tested per experiment. The error bars represent the standard
error in each experiment.
The difference in lariat ether activity observed at 20 µM and 50 µM prompted us to
examine the concentration dependence of C (5) and C10 (6) lariat ethers on primary
8
root length and lateral root density. The lateral root densities observed for C lariat at
8
8

concentrations of 5, 15, 25, 35, 45, and 50 µM were, 0.16, 0.20, 0.42, 0.62, 0.80, 1.17,
and 1.43, respectively. For the C10 lariat ether at the same concentrations, the lateral
root densities were 0.56, 0.77, 1.16, 1.20, 1.40, and 1.77. The zero concentration point
serves as a control and includes 0.2% (v/v) of DMSO in the PNS media. The increase in
lateral root density over this concentration range is essentially linear (Supplementary
2
2
Figure 1) for both compounds (R = 0.96 for 5; R = 0.95 for 6).
Assessment of lariat ether effects on A. thaliana germination and plant
growth. As observed in the hydraphile study (see Figure 2), no change in seed
germination was observed over time in the presence of 50 µM C
10 lariat ether (6), 50 µM C lariat ether amide (10) and 50 µM C10 lariat ether amide
11) (Supplementary Figure 2). These observations comport with the hypothesis that
8
lariat ether (5), 50 µM
C
8
(
both hydraphiles and lariat ethers affect plant root development and not germination.
Figure 5 shows a comparison of A. thaliana growth in the presence of active lariat ethers
5
and 6, IAA, and 2,4-D. Di-n-octyl lariat ether ([5] = 20 µM) decreased primary root
length by 76% and increased lateral root density to 0.67 (4-fold). At 20 µM, A. thaliana
plants in the presence of C lariat ether, 5, appeared on visual inspection to be healthier
than in the presence of C10 lariat ether, 6. No discoloration or chlorosis of the leaves was
observed. At a concentration of 50 µM of C (5) and C10 (6) lariat ethers, plant growth
8
8
was stunted and appeared to be similar to that observed when 1 µM 2,4-D was present.
Figure 5. Comparison of plant development in the presence of IAA (14)
and 2,4-D (15) (top row) and C
8
lariat ether (5), C10 lariat ether (6)
(
bottom row). Plants were grown in a Petri dish containing PNS media in
the presence of 5, 6, 14 or 15.
9

Effect of lariat ether amides on the growth and morphology of A. thaliana
roots. The side arms of compounds 9-13 have the same number of carbon atoms as 4-
8
, but both linkages to diaza-18-crown-6 in the former group are amidic. Amides are
inherently less flexible than the corresponding amines. [44] The macroring nitrogen
atoms of lariat ether amides are poorer donors that the nitrogen atoms of the
corresponding alkyl diazalariat ethers. [45] Any dependence of A. thaliana primary root
length on crown-ether cation binding, rather than transport, would be expected to show
results distinct from those observed for lariat ethers or hydraphiles. Figure 6 compares
the effect of lariat ethers (circles) to that of lariat ether amides (squares) at 50 µM
concentration, on primary root length. Each data point is the average of 2 trials; each
trial involved 18-23 plants. Data for compounds 4-8 may be found in Figure 3. No effect
on lateral root density was observed in A. thaliana when 9-13 were present in PNS
media at 50 µM.
Figure 6. The change in primary root length for lariat ethers (circles, 4-
8
) and lariat ether amides (squares, 9-13) at a concentration of 50 µM.
The C10 lariat ether amine (6) and amide (11) had a similar effect on the A. thaliana
primary root length. These compounds decreased the primary root length by ~95 %.
Surprisingly, 6 increased the lateral root density by ~9-fold but no change was observed
with the amide (11).
Probe of macrocyle effect on the auxin pathway. The plant hormones IAA and
2
,4-D affect both growth and ion transport. The similarity in overall effect of C and C10
8
lariat ethers to IAA and 2,4-D on A. thaliana root architecture suggested that lariat
ethers might be involved in the auxin signaling pathway. To evaluate this hypothesis, we
tested C10 lariat ether against two auxin resistant A. thaliana mutants: aux 1-7 [46] and
axr 1-12. [47] In aux 1-7, a mutated permease-like Aux1 protein significantly reduces the
rate of carrier mediated auxin uptake. [48] Axr1 encodes a mutated subunit of a
heterodimeric RUB (related to ubiquitin) activating enzyme that is unable to degrade
the Aux/IAA transcriptional repressors. Both aux 1-7 and axr 1-12 have defects in root
gravitropism and lateral root formation. Lateral root densities of Col-0, aux 1-7 and axr
1-12 plants were determined in the presence and absence of 50 ꢀM C10 lariat ether (6),
2
00 µM IAA and 200 µM 2,4-D . The results are recorded in Table 2.
1
0

Table 2. Assessment of auxin properties in genetically
modified A. thaliana plants
Primary
root length lateral roots density
mm)
Number of
Lateral root
(
Col-0 (wild type)
PNS
49.2 ± 2.2 7.4 ± 0.8
0.15 ± 0.01
0.51 ± 0.05
1.3 ± 0.1
2
00 nM IAA
00 nM 2,4-D
0 µM C10 LE (6)
7.3 ± 0.7
3.1 ± 0.2
1.5 ± 0.1
3.3 ± 0.2
3.7 ± 0.3
1.6 ± 0.2
2
5
1.2 ± 0.2
Aux 1-7
57.3 ± 1.6
PNS
5.4 ± 0.5
0.1 ± 0.01
2
00 nM IAA
00 nM 2,4-D
0 µM C10 LE (6)
28.2 ± 0.9 4.6 ± 0.5
23.8 ± 1.6 17.8 ± 1.7
0.16 ± 0.01
0.74 ± 0.06
1.43 ± 0.14
2
5
1.6 ± 0.2
2.3 ± 0.3
Axr 1-12
PNS
57.7 ± 1.4
14.1 ± 0.8
0.78 ± 0.3
0.92 ± 0.2
0.01 ± 0.05
0.07 ± 0.02
0.08 ± 0.01
1.38 ± 0.17
2
00 nM IAA
00 nM 2,4-D
0 µM C10 LE (6)
2
27.3 ± 0.9 2.1 ± 0.4
1.3 ± 0.1 1.6 ± 0.1
5
The important observations from table 2 are as follows. In the genetically modified aux
-7 and axr 1-12 mutants, similar effects to those apparent in Col-0 were observed when
1
either of the known auxins IAA or 2,4-D was present. The changes in the primary root
lengths and lateral root densities, in the presence of IAA or 2,4-D, were much smaller
for mutants. This contrasts dramatically with the presence of 6. The effect of C10 lariat
ether (6) on the root development was the same in the wild type Col-0, the auxin
transport mutant (aux 1-7), and transcriptional repressor mutant (axr 1-12). Thus, C10
lariat ether is not recognized in the auxin uptake pathway nor does it affect the
transcription repressor of AUX/IAA. It is well known that primary and lateral root
development is modulated by various molecular pathways and environment signals.
[
49] Lariat ethers may affect such molecular pathways as auxin biosynthesis in addition
to or instead of auxin transport or transcriptional repression. More likely, these
ionophores alter potassium homeostasis in root cells, which in turn, is known to alter
auxin response and root development. [50]
Pore formation by lariat ethers and lariat ether amides. Several reports of
crown ethers affecting potassium concentration or transport in plants appeared in the
1
980s. [18] [19] [20] We therefore hypothesized that the diazalariat ethers transport
potassium ions through plant root cells, which results in downstream changes in either
an auxin dependent or independent pathway. Having dispelled the auxin dependent
1
1

pathway hypothesis, the similarity in effect on plants between hydraphiles and lariat
ethers led us to query the possibility that lariat ethers were behaving in a fashion similar
to hydraphiles in membranes. Very recent results have suggested that at least some
lariat ethers may stack and form viable pores. In a study by Barboiu, Fyles, and their
coworkers, an H-bond, urea-based network led to a stacked channel. More recently, Liu
and coworkers have reported pore formation from benzo-18-crown-6-derived lariat
ethers that incorporate various switching elements.
In our own recent work, [51] we found that co-administration of either C or C11 lariat
8
ether with the antibiotics rifampicin or tetracycline showed enhanced potency against
the DH5α strain of E. coli. These experiments were done by determining the minimum
inhibitory concentration (MIC) of the lariat ether and then using concentrations of it in
1
2
the non-toxic ranges of ~ /
6
to / [MIC]. The enhancements observed ranged from 4-
3
fold to 48-fold compared to the antimicrobial toxicity in the absence of lariat ethers.
The dialkyl lariat ethers that are the focus of this report lack hydrogen bonding or
stacking elements and were not expected to form stable pores. The surprising behavior
of lariats that we observed is shown in the traces below, and parallels that results of the
studies noted above. The data shown in Figure 7 were obtained by planar bilayer voltage
clamp studies. We have recently reported planar bilayer conductance data for both C
and C11 lariat ethers. [51] We also noted there that C10 lariat showed activity, but that C12
did not. Figure 7 also shows traces for C10 lariat ether and for C and C10 lariat ether
8
8
amides. The latter could potentially form a hydrogen bonded stack leading to pores. To
our knowledge, though, there has been no previous report of pore formation by dialkyl
lariat ether amides.
Figure 7. Planar bilayer conductance (BLM) study of C
ethers and C (10) and C10 (11) lariat ether amides in asolectin
membranes. (a) The C lariat ether (4) trace showed only membrane
6
(4), C10 (6) lariat
8
6
disruption at 7 µM and 50 mV potential (scale: 10pA, 2 s). (b) The C10
lariat ether trace was obtained at a concentration of 12 µM and an applied
potential of 50 mV (scale: 0.5 pA, 2 s). This was observed at all
1
2

concentrations of 4 and applied potentials surveyed. (c) The C lariat
8
amide (10) was determined at 3 µM concentration and 30 mV applied
potential (scale: 0.5 pA, 3 s). (d) The C10 lariat ether amide (11) trace was
obtained at 7 µM and 50 mV (scale: 1 pA, 12 s). Membrane: soybean
asolectin.
Figures 3 and 4 show that among the lariat ethers surveyed, C
most active and C (4) shows no effect either on lateral root density or primary root
length. The lariat ether having C side arms (4, top panel of Figure 7), showed only
membrane disruption at all concentrations and applied potentials studied. The applied
potential for C and C10 in both cases was 50 mV but the concentrations for 5 and 6 were
µM and 12 µM, respectively. The greater number of multiple channel openings
8
(5) and C10 (6) are the
6
6
8
3
apparent for C10 may reflect this higher concentration, at least in part. In either event,
the openings are regular and continuous channel behavior was sustained for up to an
hour. The trace shown for C10 represents only 15 s. The amide traces illustrate longer
durations: C
8
~60 s and C10 ~100 s.
Three major conductance states (in picoSiemens, pS) were observed for C
8
(5) lariat
ether: 11 pS, 17 pS, and 23 pS. [51] Multiple openings are observed for these states. We
infer that C10 lariat ether is even more active as a pore-former because the multiple
openings are more numerous. The two principal open states for 6 are 15 pS and 20 pS.
The chain length dependence observed for lariat ether activity with A. thaliana
paralleled previous results obtained with bacteria. [18]
Since the presence of amide chains, R-CO<N18N>CO-R, in 9-13 reduce the donicity of
nitrogen [31] and the flexibility of the overall compound, we anticipated poorer
biological activity. [45] In contrast, C
on A. thaliana, but C -CO<N18N>CO-C
analog lariat 6. The ability of 11 to form stable pores was not expected. Surprisingly,
both the C and C10 amides formed stable, ion-conducting pores in soybean asolectin
membranes.
7
-CO<N18N>CO-C
7
(10) showed a modest effect
9
9
(11) was as biologically active as the saturated
8
Figure 7 (above) shows the traces obtained from a planar bilayer voltage clamp (BLM)
study of C (10, top) and C10 lariat (11, bottom) amides. The data for C lariat were
8
8
obtained at a concentration of 3 µM and an applied potential of 30 mV. The
concentration and potential for C10 lariat amide were 7 µM and 50 mV. The traces
shown span approximately 1 min (top) and 3.5 min (bottom). These pores remained
stable within the bilayer for an hour or more, suggesting substantial stability. Both
traces show unmistakable open-close behavior and multiple channel openings. Although
both of these compounds are biologically active in A. thaliana, plant boundary layers
and extracted asolectin bilayers are sufficiently different to preclude attempting to
directly correlate activity with pore formation. Notwithstanding, it seems clear that both
+
1
0 and 11 can form pores, which presumable affect K transport in the plants.
Both 10 (C
8
) and 11 (C10) exhibit two stable conductance states. For C lariat amide 10,
8
they are 14 and 21 picoSiemens (pS), respectively. For C10 lariat amide 11, they are 20
and 24 pS. Multiple openings are apparent in both cases. Studies of ion binding by
lariats such as C10 (6) with its counterpart diamide (11) show much reduced ion binding.
Strong binding is presumably inimical to rapid ion transport so the presence of amides
is not problematic. Indeed, the lariat ethers may well be protonated in the biological
milieu and the positive charges within the macrocycle would likely impede ion
1
3

transport. Even so, the recorded conductances are similar in both cases.
Conclusion
The present study examines the effect of known ion transporters – hydraphiles and
lariat ethers – on the growth, development, and morphology of A. thaliana. Several of
the compounds studied engendered significant morphological changes, notably in
primary root length and lateral root density. The initial hypothesis that variations in
ionophore complexation strength or transport rates could account for the effects was not
supported. The effect of these ionophores was auxin-like, but a study of two genetically
modified A. thaliana plants showed that the mechanism of action was not based on
auxin uptake or transcription repressor of auxin pathways.
Disruption of cation gradients in plant root cells is also known to affect plant growth and
development. Indeed, auxin induced growth is dependent both on extracellular
potassium ion gradients and the presence of potassium channels in plant plasma
membranes. Stress signaling caused by these synthetic amphiphiles could also affect
root development. [52] This may lead to the failure of cell elongation and root
+
development. In addition, at least five K transport proteins have been characterized in
+
A. thaliana roots. These channel proteins regulate K uptake or efflux in the root cap,
the epidermis of the primary and lateral roots, the root tip, or the root-hypocotyl
junction. Hirch, et al. reported [53] that plant nutrition and growth depends on the
AKT1 channel, which functions as an inward rectifying potassium channel. Thus, a
range of proteins could be affected by synthetic ionophore-altered cation gradients. We
speculate that the auxin signaling cascade induced by potassium channels may be
disrupted by the synthetic ionophores studied here. Alternately, the synthetic
ionophores reported here may interfere with the regulation of existing protein channels.
Lariat ethers and lariat ether amides affect the growth of A. thaliana and, surprisingly,
show clear evidence for well-behaved pore formation in soybean asolectin membranes.
The biological effects observed are similar to those of herbicide 2,4-D, which has been
hypothesized to act as a potassium carrier and increase ion transport. [54] Very recently,
silver nanoparticles were reported to show an effect similar to that of hydraphiles and
lariat ethers on A. thaliana primary root length. This change in root morphology by
silver nanoparticles was related to pore formation in plant membranes (56 ± 9 pS). [52]
Alternatively, these ionophores (hydraphiles and lariat ether) may function in an auxin
independent pathway. Alkamides such as N-isobutyldecanamide are known to decrease
primary root length and to increase lateral root density at high concentrations. [55]
Synthetic ionophores were effective against auxin transport and transcription repression
mutants. This may suggest a simple alkamide-like auxin-independent pathway. In any
event, the pore forming ability of these compounds is unprecedented and will no doubt
require extensive future study.
Acknowledgment. We thank the National Science Foundation for a grant (CHE-
1307324) that, in part, supported this work. We are also grateful to Dr. Bethany Zolman
and Gretchen Spiess for valuable discussions and technical assistance.
1
4

Experimental Section
Plant nutrient media and plating seeds. A. thaliana Columbia-0 (Col-0) seeds
(
>20), the most common strain of this plant, were sterilized and plated on plant
nutrient and 0.5% sucrose (PNS) media [56] [57] containing 0.6% agar. To make 500
mL PN media, 485 mL doubly distilled H O was first added to an autoclave beaker. The
following salts and nutrients were added to a beaker and mixed well: 5 mL of 250 mM of
KPO (pH 5.5), 2.5 mL of 1M KNO , 1 mL of 1M MgSO , 1 mL of 1M Ca(NO , 0.5 mL of
2
4
3
4
)
3 2
Ferric EDTA (1000×) and 0.5 mL of Micronutrients (1000×). [49] [50] In a 1 L
autoclaved bottle, 0.6% (3 g) of bacteriological agar was added, followed by 500 mL
solution prepared above. The media was autoclaved using the Liquid-30 program. 5 mL
of 50% sterile sucrose was added to PN media (55 °C) to make PNS media containing
0
.5% sucrose.
The types of plates, compounds, and concentrations to be used were confirmed while
the media cooled to 55 °C. Sterile centrifuge tubes (50 mL) were used to measure 29.94
mL PNS media. All the compounds were dissolved in DMSO and 60 µL of its desired
concentration was added to the PNS media, mixed by inverting the tube and poured in
the plate. IAA (Sigma-Aldrich) and 2,4-D (Sigma-Aldrich) were dissolved in autoclaved
MilliQ water. For DMSO control 60 µL DMSO was added to PNS media before it
solidified. For PNS control, 30 mL of PNS media was used. Plates were incubated at
room temperature for 2 hours before plating seeds on them.
Seeds were sterilized before plating them on the PNS media. A. thaliana Col-0 seeds
were added to sterile 1.5 mL micro-centrifuge tube. Sterilizing solution (500 µL, 30%
bleach + 0.01% Triton X-100) was added to the seeds, vortexed for 3 seconds and
incubated at room temperature for 8 minutes. After 8 minutes, sterilizing solution was
removed and 1 mL autoclaved MilliQ water was added. Seeds were briefly vortexed,
allowed to settle, and the water was removed. The seeds were washed two more times
with autoclaved MilliQ water. Seeds were finally suspended in 200 µL of sterile 0.1%
agar. These seeds were plated (> 20 seeds per plate) on PNS media using sterilized
Pasteur pipettes. Seeds were not allowed to touch each other or the boundaries of the
plates. The plates were covered with surgical tape and incubated using an Intellus
Environment Controller, under continuous white light at 23 °C. IAA was incubated for
the same time and temperature under yellow light. [58]
Germination measurements. Col-0 seeds were plated on PNS media containing
compounds 1-13 and incubated using an Intellus Environment Controller at 23 °C. The
number of Col-0 seeds germinated were observed under dissecting microscope for all
the plates. Here, radicle protrusion was scored every 12 hours for 3 days. Percentage of
seeds germinated was calculated for every reading and graphed as percent of seeds
germinated vs. time (in hours). The plates were returned to the incubator for root
development.
Primary root length measurements and number of lateral roots. A. thaliana
Col-0 seeds were incubated under white light (except IAA) for 11 days. At 11 days, the
agar around each plant was disturbed and the plant was removed from the agar using
sterile forceps. The primary root length for each plant was measured (data reported in
millimeters) using a ruler. The plant was then transferred to a petri dish containing
1
5

water, and examined by using a dissecting microscope. All the lateral roots, including
emerging and developed roots, were counted. The lateral roots emerging from the
primary root were recorded. The average and standard errors (used for error bars) for 3-
4
trials (~16-23 seeds each) was calculated. Each root length and lateral root density
value reported here is the average of values observed for ~60 plants.
Synthesis of lariat ether and lariat ether amides.
Dibenzyl hydraphile C , 1, was prepared as described in reference 34.
8
Dibenzyl hydraphile C14, 2, was prepared as described in reference 34.
Dibenzyl hydraphile C16, 3, was prepared as described in reference 34.
N,N’-Di-n-hexyl-4,13-diaza-18-crown-6, 4, was prepared in a fashion identical to
that described for 5, as described below. The product was isolated in 91% yield.
N,N’-Di-n-octyl-4,13-diaza-18-crown-6, 5, was prepared as previously reported17
except that the product was crystallization from acetone (yield: 87%). The crystals
gradually became a colorless oil on standing at room temperature.
N,N’-Di-n-decyl-4,13-diaza-18-crown-6, 6, was prepared as reported previously,17
except that the product was purified by crystallization from acetone (yield: 86%).
N,N’-Di-n-dodecyl-4,13-diaza-18-crown-6, 7, was prepared (yield: 82%) as
reported in detail in reference 17.
N,N’-Di-n-tetradecyl-4,13-diaza-18-crown-6, 8, was obtained in 83% yield after
alkylation of N,N’-diaza-18-crown-6 with 1-bromotetradecane by using the general
procedure reported in reference 17, mp 54 °C.
N,N’-Di-1-oxohexyl-4,13-diaza-18-crown-6, 9, was prepared by the reaction of
hexanoic acid with thionyl chloride, followed by reaction with 4,13-diaza-18-crown-6 as
described for 7 in reference 41. The product was isolated in 91% yield as a colorless
solid.
N,N’-Di-1-oxooctyl-4,13-diaza-18-crown-6, 10, was prepared by the reaction of
hexanoic acid with thionyl chloride, followed by reaction with 4,13-diaza-18-crown-6 as
described for 7 in reference 47. The product was isolated in 90% yield as a white solid.
N,N’-Di-1-oxodecyl-4,13-diaza-18-crown-6, 11, was prepared (yield: 92%) as
reported in reference 17.
N,N’-Di-1-oxododecyl-4,13-diaza-18-crown-6, 12, was prepared (yield 92%) as
reported in reference 17.
N,N’-Di-1-oxotetradecyl-4,13-diaza-18-crown-6, 13, was prepared by the reaction
of hexanoic acid with thionyl chloride, followed by reaction with 4,13-diaza-18-crown-6
1
6

as described for 7 in reference 47. The product was isolated in 91% yield as a white solid.
Indoleacetic acid, IAA, 14, and 2,4-Dichlorophenoxyacetic acid, 2,4-D, 15, were
purchased from Sigma-Aldrich and used as received.
Planar bilayer voltage clamp studies. Planar lipid bilayer studies were conducted
by using a Warner BC-525D bilayer clamp apparatus. The membrane was formed by
painting the 200 µM orifice with a n-decane solution of asolectin (from soybean) in n-
-1
decane (25 mg mL ). This membrane blocks theonly connection between two buffer-
filled compartments containing KCl (450 mM) buffer solution (HEPES, 10 mM, pH 7).
The lariat ether was dissolved in DMSO and added to the one side of the membrane to
achieve the desired concentration. The records of current flow at the specified potentials
were filtered with a 4-pole Bessel filter (100 Hz) and digitized at a 1 kHz sampling
interval per signal by using Clampex 9.2 (Axon instruments). Data analysis was
performed with Clampfit 9.2 (Axon instruments).
1
7

References:
1
Pedersen, C. J., Cyclic polyether and their complexes with metal salts, J. Am.
Chem. Soc. 1967, 89, 2495-2496.
2
3
4
Pedersen, C. J., Cyclic polyether and their complexes with metal salts, J. Am.
Chem. Soc. 1967, 89, 7017-7036.
Gokel, G. W.; Korzeniowsi, S. H., Macrocyclic polyether syntheses, Springer-
Verlag, 1982, 410 pp.
Gokel, G. W.; Leevy, W. M.; Weber, M., Crown ethers: Sensors for ions and
molecular scaffolds for materials and biological models, Chem. Rev. 2004, 104,
2
723-2750.
5
6
Inoue, Y.; Gokel, G. W., Cation Binding by Macrocycles, Marcel Dekker, New
York, 1990, 761 pp.
Lamb, J. D.; Christensen, J. J.; Izatt, S. R.; Bedke, K.; Astin, M. S.; Izatt, R. M.,
Effects of salt concentration and anion on the rate of carrier-facilitated transport
of metal cations through bulk liquid membranes containing crown ethers, J. Am.
Chem. Soc. 1980, 102, 3399-3403.
7
Cox, B. G.; Schneider, H. Coordination and Transport Properties of Macrocyclic
Compounds in Solution; Elsevier: Amsterdam, 1992, 420 pp.
8
Naumowicz, M.; Petelska, A. D.; Figaszewski, Z. A.; The effect of the presence of
crown ether on ion transport across the lipid bilayer, Cell Mol. Biol. Lett. 2003,
8
, 383–389.
Hernandez, J. C.; Trafton, J. E.; Gokel, G. W., Tetrahedron Lett 1991:6269–
272.
9
6
1
0
Tso, W.-W.; Fung, W.-P., Correlation between the antibacterial and alkali metal
ion transport efficiency of crown ether., Inorg. Chim. Acta 1981, 55, 129-34.
(a) Kato, N.; Ikeda, I.; Okahara, M.; Shibasaki, I., Antimicrobial activity of alkyl
crown ethers, Res. Soc. Antibac. Antifung. Agents Jpn. (Bokin Bobai) 1980, 8,
11
532-533. (b) Kato, N.; Ikeda, I.; Okahara, M.; Shibasaki, I., Antimicrobial activity
of N-alkyl monoaza crown ethers, Bokin Bobai 1980, 8, 415-420. (c) Kato, N.,
Antibacterial action of alkyl-substituted crown ethers., Kenkyu Kiyo - Konan
Joshi Daigaku 1985, 585-96. (d) Konup, L. A.; Konup, I. P.; V. E. Sklyar;
Kosenko, K. N.; V. P. Gorodnyuk; Fedorova, G. V.; Nazarov, E. I.; Kotlyar, S. A.,
Antimicrobial activity of aliphatic and aromatic crown ethers, Khimiko-
farmatsevticheskii Zhurnal 1989, 23, 578-583. (e) Gumus, A.; Karadeniz, S.;
Ugras, H. I.; Bulut, M.; Cakir, U.; Goren, A. C., Synthesis, Complexation, and
Biological Activity Studies of 4-Aminomethyl-7,8-dihydroxy Coumarines and
Their Crown Ether Derivatives, J. Heterocyclic Chem. 2010, 47, 1127-1133. (f)
Hayvali, Z.; Guler, H.; Ogutcu, H.; Sari, N., Novel bis-crown ethers and their
sodium complexes as antimicrobial agent: synthesis and spectroscopic
characterizations, Med. Chem. Res. 2014, 23, 3652–3661.
12
(a) Chui, J. K.; Fyles, T. M., Cyclodextrin ion channels. Org Biomol Chem 2014,
12 (22), 3622-34. (b) Barboiu, M.; Le Duc, Y.; Gilles, A.; Cazade, P. A.; Michau,
M.; Marie Legrand, Y.; van der Lee, A.; Coasne, B.; Parvizi, P.; Post, J.; Fyles, T.,
An artificial primitive mimic of the Gramicidin-A channel. Nat Commun 2014, 5,
4
142. (c) Matile, S.; Fyles, T., Transport across membranes. Acc Chem Res 2013,
46 (12), 2741-2. (d) Chui, J. K.; Fyles, T. M., Cyclodextrin ion channels. Org
Biomol Chem 2014, 12 (22), 3622-34. (d) Sun, Z.; Gilles, A.; Kocsis, I.; Legrand,
1
8

Y. M.; Petit, E.; Barboiu, M., Squalyl Crown Ether Self-Assembled Conjugates: An
Example of Highly Selective Artificial K Channels. Chemistry 2016, 22 (6), 2158-
2
164. (e) Sun, Z.; Barboiu, M.; Legrand, Y. M.; Petit, E.; Rotaru, A., Highly
Selective Artificial Cholesteryl Crown Ether K+-Channels. Angew Chem Int Ed
Engl 2016, 54 (48), 14473-7. (f) Gilles, A.; Barboiu, M.; Highly selective artificial
K+ channels: An example of selectivity-induced transmembrane potential, J. Am.
Chem. Soc., 2016, 138, 426-432. (g) Yang, R.-Y.; Bao, C.-Y.; Lin, Q.-N.; Zhu, L.-
Y., A light-regulated synthetic ion channel constructed by an azobenzene
modified hydraphile. Chinese Chem. Lett. 2015, 26, 851–856. (h) Bao, C.; Ma,
M.; Meng, F.; Lin, Q.; Zhu, L., Efficient synthetic supramolecular channels and
their light-deactivated ion transport in bilayer lipid membranes. New J. Chem.
2015, 39, 6297-6302. (i) Liu, T.; Bao, C.; Wang, H.; Fei, L.; Yang, R.; Long, Y.;
Zhu, L., Self-assembly of crown ether-based amphiphiles for constructing
synthetic ion channels: the relationship between structure and transport activity.
New J. Chem. 2014, 38, 3507-3513.
13
14
15
Steed, J. W.; Atwood, J. L., Supramolecular Chemistry, John Wiley and Sons,
Ltd., Chichester, 2009, 970 pp.
Tso, W. W.; Fung, W. P.; Tso, M. Y., Variability of crown ether toxicity, J. Inorg.
Biochem. 1981, 14, 237-244.
(a) Leevy, W. M.; Huettner, J. E.; Pajewski, R.; Schlesinger, P. H.; Gokel, G. W.,
Synthetic ion channel activity documented by electrophysiological methods in
living cells, J. Am. Chem. Soc. 2004, 126, 15747-15753. (b) Si, W.; Li, Z. T.; Hou,
J. L., Voltage-driven reversible insertion into and leaving from a lipid bilayer:
tuning transmembrane transport of artificial channels. Angew Chem Int Ed Engl
2014, 53 (18), 4578-81.
1
6
7
Pajewski, R.; Garcia-Medina, R.; Brody, S. L.; Leevy, W. M.; Schlesinger, P. H.;
Gokel, G. W., A synthetic, chloride-selective channel that alters chloride transport
in epithelial cells, Chem. Commun. 2006, 329-31.
Marjanovic, M.; Kralji, M.; Supek, F.; Frkanec, L.; Piantanida, I.; Smuc, T.;
Tusek-Bozic, L., Antitumor potential of crown ether: Structure activity
relationships, cell cycle disturbances, and cell death studies of a series of a series
of ionophores, J. Med. Chem. 2007, 50, 1007-1018.
1
1
8
9
Leevy. W. M.; Weber, M. E.; Gokel, M. R.; Hughes-Strange, G. B.; Daranciang,
D.; Ferdani, R.; Gokel, G. W., Correlation of bilayer membrane cation transport
and biological activity in alkyl-substituted lariat ethers, Org. Biomol. Chem.
2005, 3, 1647-1652.
1
Huang, D.; Wang, D.; Zhang, J.; Huang, L.; Fu, T.; Ou, H.; Que, R.; Zhang, Z.,
+
+
Effects of crown ethers on the K and Na transport of plant roots (1) Effect of
+
+
Benzo-15-crown-5 on the K and Na transport of wheat roots, J. Nanjing Uni.
Nat. Sci. 1980, 2, 33-44.
2
0
Macklon, A. E. S.; Sim, A.; Parsons, D. G.; Truter, M. R.; Wingfield, J. N., Effects
of some cyclic 'crown' polyethers on potassium uptake, efflux and transport in
excised root segments and whole seedlings, Ann. Bot. 1983, 52, 345-356.
Pemadasa, M. A., Effects of benzo-18-crown-6 on abaxial and adaxial stomatal
opening and its antagonism with abscisic acid. New Phytol. 1983, 93, 13-24.
Leevy, W.M.; Gokel, M.R.; Hughes-Strange, G.; Schlesinger, P.H.; Gokel, G.W.;
2
2
1
2
“
Structure and Medium Effects on Hydraphile Synthetic Ion Channel Toxicity to
the Bacterium E. coli,” New Journal of Chemistry, 2005, 29, 205–209.
1
9

2
2
3
4
Smith, B. A.; Daschbach, M. M.; Gammon, S. T.; Xiao, S.; Chapman, S. E.;
Hudson, C.; Suckow, M.; Piwnica-Worms, D.; Gokel, G. W.; Leevy, W. M., In vivo
cell death mediated by synthetic ion channels, Chem. Commun. 2011, 47, 7977-9.
Smith, B. A.; Gammon, S. T.; Xiao, S.; Wang, W.; Chapman, S.; McDermott, R.;
Suckow, M. A.; Johnson, J. R.; Piwnica-Worms, D.; Gokel, G. W.; Smith, B. D.;
Leevy, W. M., In Vivo Optical Imaging of Acute Tissue Damage Using a Near-
Infrared Fluorescent Zinc-Dipicolylamine Probe, Molecular Pharmaceutics
2
011, 8, 583-590.
2
2
5
6
Atkins, J. L.; Patel, M. B.; Cusumano, Z.; Gokel, G. W., Enhancement of
antimicrobial activity by synthetic ion channel synergy, Chem. Commun. 2010,
46, 8166-7.
Patel, M. B.; Stavri, A.; Curvey, N. S.; Gokel, G. W., Hydraphile synthetic ion
channels alter root architecture in Arabidopsis thaliana, Chem. Commun. 2014,
5
0, 11562-11564.
rd
2
2
7
Lloret, P.G., Lateral root initiation. In Plant Roots: The Hidden Hal. (3 edn),
Marcel Dekker, 2002, 127–156.
Casimiro, I.; Beeckman, T.; Graham, N.; Bhalerao, R.; Zhang, H.; Casero, P.;
Sandberg, G.; Bennett, M. J., Dissecting Arabidopsis lateral root development,
Trends In Plant Sci. 2003, 8, 165-171.
8
2
3
9
0
Peret, B.; Rybel, B. D.; Casimiro, I.; Benkova E.; Swarup, R.; Laplaze, L.;
Beeckman, T.; Bennett, M. J., Arabidopsis lateral root development: an emerging
story, Trends In Plant Sci. 2009, 14, 399-408.
Geiger, D.; Becker, D.; Vosloh, D.; Gambale, F.; Palme, K.; Rehers, M.;
Anschuetz, U.; Dreyer, I.; Kudla, J.; Hedrich, R., Heteromeric AtKC1•AKT1
+
channels in Arabidopsis roots facilitate growth under K -limiting conditions, J.
Biol. Chem. 2009, 284, 21288-21295.
3
3
1
Gokel, G. W., Hydraphiles: Design, Synthesis, and Analysis of a Family of
Synthetic, Cation-Conducting Channels, Chem. Commun. 2000, 1-9.
Gokel, G. W.; Dishong, D. M.; Diamond, C. J., Lariat Ethers. Synthesis and
Cation Binding of Macrocyclic Polyethers Possessing Axially Disposed Secondary
Donor Groups, J. C. S. Chem. Commun. 1980, 1053-4.
2
3
3
3
3
3
3
3
4
5
6
7
8
Gatto, V. J.; Gokel, G. W., Syntheses of calcium-selective, substituted diazacrown
ethers: a novel, one-step formation of bibracchial lariat ethers (BiBLES), J. Am.
Chem. Soc. 1984, 106, 8240-8244.
Curvey, N. S.; Luderer, S. E.; Walker, J. K.; Gokel, G. W., Improved Syntheses of
Benzyl Hydraphile Synthetic Cation-conducting Channels, Synthesis 2014, 46,
2
771-2779.
Notman, R.; Noro, M.; O'Malley, B.; Anwar, J., Molecular basis for
dimethylsulfoxide (DMSO) action on lipid membranes, J. Am. Chem. Soc. 2006,
1
28, 13982-3.
Notman, R.; den Otter, W. K.; Noro, M. G.; Briels, W. J.; Anwar, J., The
permeability enhancing mechanism of DMSO in ceramide bilayers simulated by
molecular dynamics, Biophys. J. 2007, 93, 2056-68.
Negin, S.; Gokel, M. R.; Patel, M. B.; Sedinkin, S. L.; Osborn, D. C.; Gokel, G. W.,
The Aqueous Medium-Dimethylsulfoxide Conundrum in Biological Studies, RSC-
Advances. 2015, 5, 8088–8093.
Plant Hormones and Their Role in Plant Growth and Development; Davies, P.
J., Ed.; Martinus Nijhoff: Leiden, 1987, pp 732.
2
0

3
9
Rahman, A.; Nakasone, A.; Chhun, T.; Ooura, C.; Biswas, K. K.; Uchimiya, H.;
Tsurumi, S.; Baskin, T. I.; Tanaka, A.; Oono, Y., A small acidic protein 1 (SMAP1)
mediates responses of the Arabidopsis root to the synthetic auxin 2,4-
dichlorophenoxyacetic acid, The Plant Jour., 2006, 47, 788–801.
Srinivas, G.; Lopez, C.; Klein, M., Membrane bound hydraphiles facilitate cation
translocation. J. Phys. Chem. B 2004, 108, 4231-4235.
Weber, M. E.; Schlesinger, P. H.; Gokel, G. W., Dynamic Assessment of Bilayer
Thickness by Varying Phospholipid and Hydraphile Synthetic Channel Chain
Lengths. J. Am. Chem. Soc. 2005, 126, 636-642.
4
0
1
4
4
2
Arnold, K. A.; Hernandez, J. C.; Li, C.; Mallen, J. V.; Nakano, A.; Schall, O. F.;
Trafton, J. E.; Tsesarskaja, M.; White, B. D.; Gokel, G. W., Analysis of sodium,
potassium, calcium, and ammonium cation binding selectivity in one- and two-
armed nitrogen-pivot lariat ethers, Supramol. Chem. 1995, 5, 45-60.
Gokel, G. W.; Schall, O. F. In Comprehensive Supramol. Chem.; Gokel, G. W.,
Ed.; Pergamon: Oxford, 1996; Vol. 1; pp 97-152.
Weber, M. E.; Wang, W.; Steinhardt, S. E.; Gokel, M. R.; Leevy, W. M.; Gokel, G.
W., The Influence of Varied Amide Bond Positions on Hydraphile Ion Channel
Activity, New J. Chem. 2006, 30, 177-184.
4
3
4
4
45
Gokel, G. W.; J. C. Hernandez; A. M. Viscariello; K. A. Arnold; C. F. Campana; L.
Echegoyen; F. R. Fronczek; R. D. Gandour; C. R. Morgan; J. E. Trafton; S. R.
Miller; C. Minganti; D. Eiband; R. A. Schultz; M. Tamminen, Steroidal Lariat
Ethers: A New Class of Macrocycles and the Crystal Structure of N-
(
cholesteryloxycarbonyl)aza-15-crown-5. J. Org. Chem. 1987, 52, 2963-2968.
4
6
7
Pickett, F. B.; Wilson, A. K.; Estelle, M., The aux 1 mutation of Arabidopsis
confers both auxin and ethylene resistance, Plant Physiol. 1990, 94, 1462-1466.
Dharmasiri, N.; Dharmasiri, S.; Weijers, D.; Karunarathna, N.; Jurgens, G.;
Estelle, M., AXL and AXR1 have redundant functions in RUB conjugation and
growth and development in Arabidopsis, The Plant Jour., 2007, 52, 114-123.
Marchant, A.; Kargul, J.; May, S. T.; Muller, P.; Delbarre, A.; Rechenmann, C. P.;
Bennett, M. J., Aux1 regulates root gravitropism in Arabidopsis by facilitating
auxin uptake within root apical tissues, EMBO Jour. 1999, 18, 2066-2073.
(a) Chen, H.; Xiong, L., Pyridoxine is required for post-embryonic root
development and tolerance to osmotic and oxidative stresses, The Plant J., 2005,
4
4
8
9
4
44, 396-408. (b) Linkohr, B. I.; Williamson, L. C.; Fitter, A. H.; Leyser, H. M. O.,
Nitrate and phosphate availability and distribution have different effects on root
system architecture of Arabidopsis, The Plant J., 2002, 29, 751-760. (c) Xuan,
W.; Band, L.; Kumpf, R. P.; Van Damme, D.; Parizot, B.; De Rop, G.; Opdenacker,
D.; Moller, B. K.; Skorzinski, N.; Njo, M. F.; Rybel, B.; Audernaert, D.; Nowack,
M. K.; Vanneste, S.; Beeckman, T., Cyclic programmed cell death stimulates
hormone signaling and root development in Arabidopsis, Science, 2016, 351,
3
84-387.
5
0
1
Claussen, M.; Luethe, H.; Blatt, M.; Boettger, M., Auxin-induced growth and its
linkage to potassium channels, Planta 1997, 201, 227-234.
Negin, S.; Patel, M. B.; Gokel, M. R.; Meisel, J. W.; Gokel, G. W., Antibiotic
Potency against E. coli Is Enhanced by Channel-Forming Alkyl Lariat Ethers,
Chembiochem 2016, 17, 2153-2161.
5
52
Sosan, A.; Svistunenko, D.; Straltsova, D.; Tsiurkina, K.; Smolich, I.; Lawson, T.;
Subramaniam, S.; Golovko, V.; Anderson, D.; Sokolik, A.; Colbeck, I.; Demidchik,
2
1

V., Engineered silver nanoparticles are sensed at the plasma membrane and
dramatically modify the physiology of Arabiopsis thaliana plants, The Plant J.
2
016, 85, 245-257.
5
3
4
Hirsch, R. E.; Lewis, B. D.; Spalding, E. P.; Sussman, M. R., A role for the AKT1
potassium channel in plant nutrition, Science 1998, 280, 918-21.
Smejtek, P.; Paulis-Illiangasekare, M.; Modification of ion transport in lipid
bilayer membranes in the presence of 2,4-dichlorophenoxyacetic acid, Biophys.
Jour., 1979, 26, 441-466.
5
55
Morquecho-Contreras, A.; Mendez-Bravo, A.; Pelagio-Flores, R.; Raya-Gonzalez,
J.; Ortiz-Castro, R.; Lopez-Bucio, J., Characterization of drr1, an alkamide-
resistant mutant of arabidopsis, reveals an important role for small lipid amides
in lateral root development and plant senescence; Plant Physiol., 2010, 152,
1659-1673.
5
5
5
6
Haughn, G. W.; Somerville, C., Sulfonylurea-resistant mutants of Arabidopsis
thaliana; Mol. Gen. Genet., 1986, 204, 430-434.
Gamborg, O. L.; Murashige, T.; Thorpe, A.; Vasil, I. K., Plant tissue culture
media. In Vitro 1976, 12, 473-478.
Leasure, C. D.; Chen, Y.; He, Z. H., Enhancement of indole-3-acetic acid
photodegradation by vitamin B6. Mol. Plant, 2013, 6, 1992-1995.
7
8
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Supramolecular Cation Transporters Alter Root
Morphology in the Arabidopsis Thaliana Plant
‡
¶
†¶
‡
†‡¶
Mohit B. Patel, Saeedeh Negin, Ariel Stavri, and George W. Gokel *
Highlights:
•
The synthetic ion transporters – hydraphiles and lariat ethers – engendered
significant morphological changes, notably in primary root length and lateral root
density of Arabidopsis thaliana plant.
•
•
The change in root morphology was dependent on length and concentration of
the synthetic ionophores used.
Lariat ethers and lariat ether amides affect the growth of A. thaliana and,
surprisingly, show clear evidence for well-behaved pore formation in soybean
asolectin membranes.
•
A study of two genetically modified A. thaliana plants showed that the
mechanism of action was not based on auxin uptake or transcription repressor of
auxin pathways. The change in root morphology may be associated with the pore
forming and ion transporting ability of these compounds.
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