An Activity-Based Sensing Fluorogenic Probe for Monitoring Ethylene in Living Cells and Plants

Article abstract of DOI:10.1002/anie.202108335

Ethylene (ET) is an important gaseous plant hormone. It is highly desirable to develop fluorescent probes for monitoring ethylene in living cells. We report an efficient Rh^III-catalysed coupling of N-phenoxyacetamides to ethylene in the presenc

Full text of DOI:10.1002/anie.202108335

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: An Activity-Based Sensing Fluorogenic Probe for Monitoring
Ethylene in Living Cells and Plants
Authors: Yiliang Chen, Wei Yan, Duojing Guo, Yu Li, Ji Li, Hao Liu,
Lirong Wei, Na Yu, Biao Wang, Ying Zheng, Maofeng Jing,
Jing Zhao, and Yong-Hao Ye
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202108335
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10.1002/anie.202108335
Angewandte Chemie International Edition
RESEARCH ARTICLE
An Activity-Based Sensing Fluorogenic Probe for Monitoring
Ethylene in Living Cells and Plants
Yiliang Chen,[a, b] Wei Yan,[a, b] Duojing Guo,[a, b] Yu Li,[a, b] Ji Li,[c] Hao Liu,[a, b] Lirong Wei,[a] Na Yu,[a, b]
Biao Wang,[a, b] Ying Zheng,[a] Maofeng Jing,[a] Jing Zhao,[c] Yonghao Ye*[a, b]
[a]
Prof. Dr. Y.H. Ye, Dr. Y.L. Chen, Dr. W. Yan, D.J. Guo, Y. Li, H. Liu, L.R. Wei, Prof. Dr. N. Yu, B. Wang, Y. Zheng, Prof. Dr. M.F. Jing
Key Laboratory of Plant Immunity, College of Plant Protection
Nanjing Agricultural University
Nanjing 210095, P. R. China
E-mail: yeyh@njau.edu.cn
[b]
[c]
Prof. Dr. Y.H. Ye, Dr. Y.L. Chen, Dr. W. Yan, D.J. Guo, Y. Li, H. Liu, Prof. Dr. N. Yu, B. Wang
State & Local Joint Engineering Research Center of Green Pesticide Invention and Application
Nanjing Agricultural University
Nanjing 210095, P. R. China
Dr. J. Li, Prof. Dr. J. Zhao
State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC),
School of Chemistry and Chemical Engineering
Nanjing University
Nanjing 210023, P. R. China
Supporting information for this article is given via a link at the end of the document.
Abstract: Ethylene (ET) is an important gaseous plant hormone. It is
highly desirable to develop fluorescent probes for monitoring ethylene
in living cells. Herein we report an efficient Rh(III)-catalysed coupling
of N-phenoxyacetamides to ethylene in the presence of an alcohol.
The newly discovered coupling reaction exhibited a wide scope of N-
phenoxyacetamides and excellent regioselectivity. More importantly,
we successfully develop three fluorophore-tagged Rh(III)-based
fluorogenic probes (coumarin-ethylene probes, CEPs) using this
strategy for the selective and quantitative detection of ethylene.
Among the probes, CEP-1 exhibited the highest sensitivity with a limit
of detection of ethylene at 52 ppb in air. Furthermore, CEP-1 was
successfully applied for imaging in living CHO-K1 cells and for
monitoring endogenous-induced changes in ethylene biosynthesis in
tobacco and Arabidopsis thaliana plants. These results indicated that
CEP-1 has great potential to illuminate the spatiotemporal regulation
of ethylene biosynthesis and ethylene signal transduction in living
biological systems.
responses. Although the ethylene signal transduction pathway has
been extensively studied, the biochemical modes of action of many
of the signaling components are still unresolved.[3] Therefore, the
development of a real-time monitoring method for ethylene is
helpful for the insight of ethylene biology. Unfortunately, there are
no cost-effective methods available to rapidly quantitate ethylene
concentrations.[4] Currently, the primary technologies for ethylene
detection include gas chromatography,[5] photoacoustic
spectroscopy,[6] and electrochemical methods;[7] however, these
technologies usually suffer from high costs or lacking real-time
monitoring of ethylene levels.
Fluorescent, especially fluorogenic, metal complexes have
been widely applied in complex biomolecule modification and
especially useful in detecting ethylene via its coordination or reactivity.
Silver(I) and copper(I) can reversibly coordinate ethylene. The
pioneering work in Burstyn group described a poly(vinyl phenyl
ketone) (PVPK) polymer film that luminesces when coordinated to
impregnated silver(I) ions. Upon exposure to ethylene gas, the
binding is disrupted, and the silver(I) ion mediates quenching of the
PVPK photoluminescence.[8] Later, Swager and colleagues reported
an ingenious design of a sensor where the quenching of the
fluorescence of a conjugated polymer by copper(I) complexes could
be reversed by binding ethylene.[9] Pivotal progress by the Hitomi
group described an interesting silver(I)-anthracene complex
containing an intramolecular metal-arene interaction, which could be
disrupted by exposure to ethylene gas.[10] However, these
approaches were not applied in biological systems. In situ metabolite
imaging by optical methods is becoming increasingly important in
chemical biology[11]. Activity-based sensing (ABS), which utilizes
molecular reactivity, rather than conventional lock-and-key molecular
recognition for analyte detection. Notably, Chang et al. extensively
applied this ABS strategy in the selective detection of molecular
analytes in complex biological environments, especially on metal-
dependent reactivity for bioimaging and related purposes.[12] Recently,
tremendous progress was made by Michel,[13] Huang,[14] and
Tanaka,[15] who took advantage of the well-known Grubbs catalyst
and designed highly sensitive fluorescent probes to detect ethylene
Introduction
Ethylene (ET) is an important gaseous plant hormone that
is involved in a variety of growth and development processes,
such as seed germination, cell elongation, fertilization, fruit
ripening, and seed dispersal. It also plays important roles in
pathogen defence and response to external stress factors.[1] The
ethylene biosynthesis pathway has been well established and
consists of two main steps: the ethylene precursor SAM (S-
adenosyl-L-methionine) is first converted to ACC (1-
aminocyclopropane-1-carboxylic acid) by ACC synthase (ACS),
and then ACC is oxidized by ACC oxidase (ACO) to generate
ethylene.[2] The canonical signaling pathway is that ethylene
signaling involves ethylene receptors (ETR1, ERS1, ETR2, EIN4,
ERS2), the protein kinase CTR1, and EIN2 that signals to the
transcription factors EIN3, EIL1, and EIL2. These, in turn, signal to
other transcription factors such as the ERFs leading to ethylene
1
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10.1002/anie.202108335
Angewandte Chemie International Edition
RESEARCH ARTICLE
in vivo by olefin-metathesis. These ruthenium-based strategies
tremendously improved the potential of the applications in more
complex biological systems. Inspired by these previous work, we aim
to design a fluorogenic probe for the selective detection of
endogenous ethylene with high sensitivity and to explore other metals
(besides silver, copper, ruthenium).
In 2014, Zhao group reported Rh(III) catalysed the reaction
of N-phenoxyacetamides with α,β-unsaturated aldehydes to
synthesize 1,2-oxazepines through the activation of C-H/[4 + 3]
annulation.[16] Another report from Zhao group indicated that the
use of Rh(III) to catalyse the tandem reaction of N-
phenoxyacetamides and alkynes with multiple functional groups
led to structural diversity.[17] In 2017, Zhao group described a
the role of intermediate A in the hydrocarbon activation reaction
with alkene as the substrate, we designed a fluorophore-tagged
rhodium metal complex that might provide a selective and
sensitive chemodosimeter for the detection of ethylene. Herein,
Rh(III) metal catalysts acted as quenchers to bind coumarin to
form the nonfluorescent CEP (coumarin-ethylene-probe), and
reaction with ethylene gas formed CEP-ET, resulting in the
fluorescence “turn-on” of coumarin. Coumarin-metal complexes
were rationally designed and synthesized as
a real-time
fluorescent sensor to detect ethylene gas in CHO-K1 cells,
tobacco and Arabidopsis leaves.
Rh(III)-catalysed
ortho-arylation
reaction
of
N-
Results and Discussion
phenoxyacetamides with benzothiazoles to obtain corresponding
mono/disubstituted arylated phenol products.[18] These
hydrocarbon activation reactions can produce the same
intermediate A, a Rh(III)-based five-membered ring (Scheme 1),
We first performed optimization experiments on the
hydrocarbon activation reaction of N-phenoxyacetamide (1a) with
ethylene. Encouragingly, the treatment of 1a with ethylene in the
presence of [Cp*RhCl2]2 (10 mol %) and CsOAc (1.0 equiv) in
various alcohols (MeOH, EtOH, n-propanol, ethylene glycol) at
room temperature for 5 min led to the formation of the catenarian
and intermediate
A could react with various unsaturated
hydrocarbons to form the corresponding products. Given that
ethylene gas is the simplest alkene, we hypothesized that N-
phenoxyacetamides could also react with ethylene and carried
out exploring experiments to test this hypothesis, which led the
discovery of a novel type of ethylene activation reaction. The
reaction mechanism indicates that intermediate A could couple
with ethylene and an alcohol to form new C-H bonds. Inspired by
products
2-(1-methoxyethyl)phenol
(2aa,
87%),
2-(1-
ethoxyethyl)phenol (2ab, 79%), 2-(1-propoxyethyl)phenol (2ac,
83%) and the annulated product 5-methyl-2,3-dihydro-5H-
benzo[e][1,4]dioxepine (2ad, 68%) (Table 1, entries 9, 11-13).
Compared to [Cp*RhCl2]2, other transition metal catalysts, such as
iridium, ruthenium, and platinum, are ineffective under current
conditions (Table 1, entries 1-4). The replacement of CsOAc with
other additives resulted in a slightly lower yield (Table 1, entries
5-7). Further investigation of solvents revealed that the reaction
could be carried out only with the participation of alcohol solvents,
while DCM, THF, 1,4-dioxane, and PBS buffer (PB) essentially
gave no product formation (Table 1, entries 14-17).
Table 1. Optimization of Reaction Conditions.
Additive
(equiv)
Entry
Catalyst
Solvent
% Yield
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
[Cp*IrCl2]2
[Ru(pcymene)Cl2]2
Ru(COD)Cl2
[Pt(NH3)2Cl2]
[Cp*RhCl2]2
[Cp*RhCl2]2
[Cp*RhCl2]2
[Cp*RhCl2]2
[Cp*RhCl2]2
[Cp*RhCl2]2
[Cp*RhCl2]2
[Cp*RhCl2]2
[Cp*RhCl2]2
[Cp*RhCl2]2
[Cp*RhCl2]2
[Cp*RhCl2]2
[Cp*RhCl2]2
[Cp*RhCl2]2
[Cp*RhCl2]2
CsOAc (1.0)
CsOAc (1.0)
CsOAc (1.0)
CsOAc (1.0)
K2CO3 (1.0)
Na2CO3 (1.0)
CsOPiv (1.0)
CsOAc (0.5)
CsOAc (1.0)
CsOAc (2.0)
CsOAc (1.0)
CsOAc (1.0)
CsOAc (1.0)
CsOAc (1.0)
CsOAc (1.0)
CsOAc (1.0)
CsOAc (1.0)
CsOAc (1.0)
CsOAc (1.0)
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
EtOH
n-propanol
EG
DCM
THF
1,4-Dioxane
PB
ND
ND
ND
ND
2aa (68)
2aa (65)
2aa (64)
2aa (70)
2aa (87)
2aa (82)
2ab (79)
2ac (83)
2ad (68)
ND
ND
ND
ND
MeOH: PB =1:9 2aa (57)
EG: PB =1:9 2ad (63)
Scheme 1. Design of the fluorogenic detection of ethylene by a Rh(III)-coumarin
complex.
ND, not detected; EG, ethylene glycol; PBS buffer, PB; r.t., room temperature.
2
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RESEARCH ARTICLE
Under optimal conditions, the substrate scope of N-
phenoxyacetamides was first investigated. As shown in Table 2,
various N-phenoxyacetamides with ortho, meta, or para electron-
neutral and electron-donating substituents such as CH3 and
OCH3 groups (2aa-2ad, 2e, 2f, 2k, 2i, 2q, and 2r) afforded
excellent selectivity and high yields. Electron-withdrawing
functional groups (such as F, Cl, and Br) were well tolerated (2b-
2d, 2h-2j, and 2n-2p), and the corresponding products were
isolated in yields ranging from 73% to 88%. It is worth noting that
the reaction of N-phenoxyacetamide substrates containing an
electron-withdrawing trifluoromethyl group (2g, 2m, and 2s)
resulted in poor yields (52%, 47, and 48%, respectively). In
particular, the meta-substituents on N-phenoxyacetamides
possessed good regioselectivity, and functionalization occurred
mainly on the ortho C-H at the C-2 position (2h-2m).
Rh(III) cyclopentenyl complex possesses relatively high water
solubility, which supports its direct application for ethylene
sensing in complex biological systems. The preparation of these
probes is very straightforward, and the route is shown in Figure
1b.
Various solvents were evaluated for dissolving probes to
detect ethylene, including dimethyl sulfoxide (DMSO), PBS buffer
(PB), dichloromethane (DCM), ethyl acetate (EA), 1,4-dioxane
(1,4-Diox), dimethyl formamide (DMF), tetrahydrofuran (THF),
acetonitrile (ACN), methanol (CH3OH) + PB, ethanol (EtOH) + PB,
n-propanol (NPA) + PB, n-butanol (NBA) + PB, and ethylene
glycol (EG) + PB. As shown in Figure S3, adequate ethylene gas
was injected into different probe solutions, and no significant
fluorescence intensity changes were observed upon the
application of any of these organic solvents except the alcohol
solvents. However, when alcohol solvents were used as part of
the reaction solvent, the reaction system would release a stronger
fluorescent species upon exposure to ethylene gas, which
illustrated that the solvent plays an extremely important role in the
detection performance of the fluorescence probes.
Table 2. Scope of Rh-catalysed ortho-hydrocarbon activation.*
To further clarify the mechanism of the reaction between the
probe and ethylene, 1H NMR titration experiments were
performed in methanol-d4 solvent. As shown in Figure S4, the
chemical shifts at 6.16, 7.84, 7.21, 6.81, 2.21, and 1.67 ppm were
assigned to the H1~H6 protons of CEP-1, respectively. The CEP-
1 solution was injected with different volumes of ethylene gas for
2 min, and as the volume of ethylene increased, the intensity of
the hydrogen proton signals belonging to CEP-1 disappeared
gradually; meanwhile, a new set of peaks (δH1’ = 6.08 ppm, δH2’
= 7.79 ppm, δH3’ = 7.33 ppm, δH4’ = 6.74 ppm, δH5’ = 5.19 ppm,
δH6’ = 1.56 ppm) appeared, which indicated that a chemical
reaction occurred between CEP-1 and ethylene to generate new
substances. The 1H and 13C NMR spectrum of the separated
fluorescent substance also showed the transformation of CEP-1
to CEP-1-ETa (Figure S35). While performed in methanol-d4
solvent, the 1H and 13C NMR spectrum lacked a methyl peak with
chemical shift of approximately 3.14 ppm and 56.4 ppm (Figure
S36), respectively, proving that the methanol solvent not only
acted as a solvent but also participated in the reaction. In addition,
50 mM CH3OH, EtOH, n-propanol, n-butanol, and ethylene glycol
were utilized to dissolve CEP-1 and generate various fluorescent
substances to detect ethylene (Figure 1c).
2aa, 87%
2c, 78%
2ab, 79%
2d, 88%
2ac, 83%
2e, 74%
2ad, 68%
2f, 76%
2b, 78%
2g, 52%
2h, 73%
2m, 47%
2i, 74%
2n, 84%
2j, 73%
2o, 75%
2k, 83%
2p, 79%
2l, 88%
2q, 82%
2r, 66%
2s, 48%
*Reaction conditions: 0.2 mmol of 1, 5 mL of ethylene, [RhCp*Cl2]2 (10 mol%),
1.0 equiv of CsOAc in 0.2 M R1OH at room temperature for 5 min.
We further designed and synthesized three fluorophore-
tagged Rh(III)-based probes for the selective and quantitative
detection of ethylene in vivo. In this report, we chose the Rh(III)
metal complex (Moiety A Figure 1a) as the fluorescent response
site and ethylene binding site because it meets the following
requirements. First, the presence of rhodium could quench the
fluorescence of the coumarin core via heavy metal atom
electronic effects. Second, upon reacting with ethylene, a
corresponding hydrocarbon activation reaction occurs to replace
rhodium and generate a new fluorescent substance. Third, the
Figure 1. Preparation of the fluorophore-tagged Rh(III)-based probes. a)
Diagram of the design of fluorescent probes with moiety A as the fluorescent
3
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10.1002/anie.202108335
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RESEARCH ARTICLE
response site and ethylene binding site. b) Synthetic route of CEP. Reagents
and conditions: (1) NaH, O-(mesitylsulfonyl)hydroxylamine, DMF; (2) Ac2O,
DCM; (3) [Cp*RhCl2]2, K2CO3, CH3CN. c) Chemical structure of fluorescent
substance after injecting excess ethylene into CEP-1 dissolved in different
alcohol solvents (CH3OH, EtOH, n-propanol, n-butanol, and ethylene glycol)
and diagram illustrating turn-on with CEP-1 in the presence of ethylene.
illustrated in Figure 2f, the addition of ethylene (2 mL) induced a
strong fluorescence enhancement, whereas other tested
interferents did not trigger an apparent fluorescence
enhancement. This result indicated that CEP-1 displayed
excellent selectivity towards ethylene and was suitable for
detecting ethylene in biological samples.
Next, a series of CEP-1 concentration systems were
evaluated to determine the best response of CEP-1 to ethylene.
In line with the change in fluorescence intensity before and after
the addition of ethylene for 2 min to different concentrations of the
CEP-1 solution, we finally chose 100 μM as the optimal CEP-1
concentration (Figure 2a). Additionally, the absorption spectra of
CEP-1 in PBS buffer solution (100 μM, pH = 7.3, containing 10%
ethylene glycol) with ethylene were investigated. Figure 2b shows
an absorption maximum at 340 nm for the CEP-1 solution alone;
however, new absorption bands emerged at 368 nm and 328 nm
after the addition of ethylene for 2 min, indicating that a chemical
reaction occurred between CEP-1 and ethylene, which is
1
consistent with the H NMR results (Figure S4). Besides, time-
dependent reaction kinetic experiment showed that CEP-1 reacts
with ethylene very rapidly, and the fluorescence intensity reached
the maximum within 2 minutes after the addition of 2 mL ethylene
(Figure 2c).
With these findings in mind, we continued to evaluate the
ability of the probes to detect various concentrations of ethylene
gas. This was done by injecting ethylene into solutions of different
probes (100 μM, pH = 7.3, containing 10% ethylene glycol) via a
gastight syringe and measuring the fluorescence intensity after 2
min (Figure 2d, Figure S5 and S6). A good linear correlation was
observed between the fluorescence intensity at 460 nm and the
volume of ethylene in the range of 10-250 μL. The limits of
detection (LODs) for ethylene were calculated to be 0.29 μL for
CEP-1, 0.34 μL for CEP-2 and 0.49 μL for CEP-3 based on the
IUPAC definition (CDL = 3σ/k),[19] which correspond to 52 ppb, 60
ppb, and 88 ppb, respectively, in the headspace of the sample
bottle. These values are low enough to detect the <1 ppm levels
of ethylene commonly found in many biological samples during
ripening.[20] As shown in Table S1, among the three CEPs, CEP-
1 displayed the best optical properties with the highest molar
extinction coefficient (1.04 M-1 cm-1) and fluorescence quantum
yields (0.79) when reacted with ethylene. In addition, compared
with other previously published fluorophore-tagged Grubbs-
Catalyst-Based ethylene probes, CEP-1 shows obvious
advantages in detection limit, response time, selectivity and
Stokes shift, except the relatively short emission wavelength
(Table S2). Thus, we decided to utilize CEP-1 for further
investigation.
Figure 2. Spectral characterization and selectivity of CEP-1. a) Fluorescence
intensity changes of different concentrations of CEP-1 (0-200 μM, λex = 365 nm)
in the presence of excess ethylene in a mixed EG/PB (v/v = 1/9) solvent. The
inset shows the fluorescence change of CEP-1 (100 μM). b) Absorption spectra
of CEP-1 (100 μM) in the absence and presence of excess ethylene in EG/PB
(v/v = 1/9). c) Time-based monitoring of CEP-1 (100 µM) exposed to a
theoretical concentration of 2 mL of ethylene (957 mM) dissolved in EG/PB (v/v
= 1/9). λex = 365 nm/λem = 460 nm. d) Fluorescence intensity of CEP-1 (100 μM)
as a function of the ethylene gas volume in EG/PB (v/v = 1/9) solvent. λex = 365
nm/λem = 460 nm. End points represent normalized fluorescence after the
addition of ethylene for 2 min. e) pH dependence of the CEP-1-ethylene system
at different pH conditions in EG/PB (v/v = 1/9). λex = 365 nm/λem = 460 nm. f)
Selectivity study of CEP-1 (100 μM) against glutathione and various types of
terpenes (6 mM). λex= 365 nm/λem = 460 nm. The data shown are the average
of four independent experiments. Error bars denote SD.
Subsequently, the response of CEP-1 to ethylene was
detected at pH values of 2 to 12 in a mixture of EG/H2O (1/9)
solution. As shown in Figure 2e, CEP-1 (100 μM, containing 10%
ethylene glycol) itself exhibited no fluorescence in this pH range,
while after the addition of ethylene (2 mL) for 2 min, the strongest
fluorescence intensity appeared in the pH range of 6 to 12, which
was within the normal physiological range. These results
indicated that CEP-1 has potential applications for the detection
of ethylene in biological samples.
To verify the selectivity of CEP-1 for ethylene, studies were
conducted in the presence of glutathione and other competitive
unsaturated plant metabolite molecules, including farnesol,
citronellol, limonene, myrcene, pinene, and terpinolene. As
The encouraging in vitro results prompted us to determine
whether CEP-1 could be applied to visualize ethylene in live cells
using confocal fluorescence microscopy. The cytotoxicity of CEP-
1 before or after treated with ethylene was evaluated with a CCK-
8 assay, which was performed on CHO-K1 cells incubated with
CEP-1 at different concentrations (0, 1, 2, 4, 6, 8, 10 μM) for 12 h.
The CCK-8 results indicated that the cell viability of the treatments
with or without ethylene were both higher than 80% even after
incubation with 10 μM CEP-1 (EG/PB = 1/9) for 12 h, suggesting
almost negligible cytotoxicity of CEP-1 and its reaction products
to living CHO-K1 cells (Figure S42). Then, CEP-1 was tested for
its ability to visualize changes in ethylene levels in live cells. CHO-
K1 cells were incubated with 10 μM CEP-1 for 15 min at 37 °C,
4
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10.1002/anie.202108335
Angewandte Chemie International Edition
RESEARCH ARTICLE
leading to almost no fluorescence. However, when living CHO-K1
cells were treated with ethylene gas (0.5 mL and 2 mL,
respectively) for 30 min, and then incubated with CEP-1 (10 μM)
at 37 °C under 5% CO2 for another 30 min and imaged washing
with PBS, a prominent blue fluorescence response was observed
(Figure 3a). Flow cytometry was used to quantify the fluorescence
intensity of CHO-K1 cells. It is obvious that the ethylene and CEP-
1 co-treated CHO-K1 cells show much higher fluorescence
signals than only CEP-1 treated cells (Figure 3c), which is
consistent with the fluorescence intensity measured by ImageJ
software (Figure 3b). These results indicated that CEP-1 has
good cell permeability and low toxicity and can be exploited for
cell imaging and the rapid detection of ethylene in vivo.
selected ACC to enhance ethylene production. As shown in
Figure S43a, two-month-old tobacco leaves were incubated with
ACC for 12 h, and then the tobacco leaves were treated with 10
μM CEP-1 (EG/PB = 1/9) for 30 min before imaging. As quantified
in Figure S43b, no fluorescence enhancement was observed in
tobacco leaves incubated with deionized water or ACC (1 mM)
only. The tobacco leaves incubated with the CEP-1 (10 μM) had
weak fluorescence, suggesting that CEP-1 could detect basal-
level ethylene produced in tobacco leaves. In addition, a large
fluorescence enhancement was observed when the tobacco
leaves were pretreated with ACC for 12 h followed by treatment
with CEP-1 for 30 min.
To further confirm the applicability of CEP-1 in detecting
ethylene in the plant samples, the model plant Arabidopsis
thaliana was tested. As shown in Figure 4a, leaf epidermal peels
from old A. thaliana plants were subjected to local treatment with
ethylene precursor (ACC) for plant ethylene accumulation. As
quantified in Figure 4b, the levels of ethylene produced in the
epidermal peels via ACC treatment were significantly higher than
those produced in untreated leaf peels of A. thaliana. In addition,
we used CEP-1 to detect time-dependent ethylene production in
leaf peels of A. thaliana (Col-0) plants subjected to a 12 h
stimulant (ACC) treatment. Confocal microscopy imaging shows
that leaf peels fluorescence increased in a time-dependent
manner within 30 minutes after treated with CEP-1 (Figure 4c and
4d). These data are consistent with literature reports that have
shown consistently triggered ethylene gas production in Col-0
plants after ACC exposure.[21]
To demonstrate the sensitivity of CEP-1 to detect
endogenous differences in ethylene production, we applied our
strategy to measure systemic ethylene gas accumulation in two
Figure 3. Visualization of ethylene in live cells using CEP-1. a) Confocal
microscopy images of ethylene detection in live CHO-K1 cells incubated with 10
μM CEP-1 for 30 min in the (A) absence and (B and C) presence of ethylene
(injected with 0, 0.5, 2 mL of ethylene for 30 min): (1) brightfield; (2) bule
channel; (3) merged. Scale bars, 100 µm. The images were collected at 410-
480 nm (blue channel) upon excitation at 405 nm. b) Normalized mean
fluorescence intensities extracted from ROIs using the ImageJ software. Plotted
data are the average of three independent experiments. Each experiment was
repeated for three times. Three fields (290.62 μm × 290.62 μm, 15 cells of each
field) of view per replicate were measured. Error bars denote ± SD. Statistical
analysis was performed using one-way ANOVA with Tukey’s multiple
comparisons test. **P < 0.002, ns = not significant. c) Flow cytometry analysis
of CHO-K1 cells were pre-treated with 0, 0.5, 2 mL of ethylene for 30 min and
then incubated with 10 μM CEP-1 for 30 min. Set the excitation wavelength at
405 nm and collect the fluorescence signals at 445/45 nm.
mutants, acs2/6/4/5/9 and 4myc-ACS6DDD
.
The mutant
acs2/6/4/5/9, which contains knock outs of five functional ACS
genes, produced much lower levels of ethylene during pathogen
infection.[22] The mutant 4myc-ACS6DDD is a phosphor-mimicking
form.[23] Compared with wild-type Col-0, a knockout acs2/6/4/5/9
that has a reduced capacity to generate ethylene during plant
immune response, showed no significant fluorescent change after
CEP-1 addition (Figure 5a and 5c). In contrast, the phosphor-
mimicking 4myc-ACS6DDD exhibited a significant increase in
fluorescence compared to wild-type Col-0 (Figure 5b and 5c).
These findings demonstrated that the measured endogenous
ethylene levels are directly linked to endogenous ethylene
production and that the developed method could be used to
characterize different mutants with altered ethylene levels.[22, 24]
We further investigated the capacity of CEP-1 to detect
changes in endogenous ethylene levels in live plant samples.
Considering that ACC is the immediate precursor of ethylene and
is often used to elevate ethylene biosynthesis in plant, we
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Figure 4. Detection of ethylene generated in A. thaliana plants. a) Fluorescence and brightfield imaging of ethylene detection in A. thaliana plants subjected to a 12
h stimulant (ACC) treatment. Scale bars, 100 µm. b) Normalized mean fluorescence intensities of A. thaliana plants with ACC treatment. c) Time-dependent detection
of ethylene in A. thaliana plants subjected to a 12 h stimulant (ACC) treatment. Scale bars, 100 µm. d) Normalized mean fluorescence intensities of time-dependent
detection of ethylene in A. thaliana plants with ACC treatment. Plotted data are the average of three independent experiments. Each experiment was repeated for
three times. Mean fluorescent intensity was measured from five individual leaf peels (fifteen regions in random, 290.62 μm × 290.62 μm of each region) using
ImageJ software. Error bars denote ± SD. ***P < 0.0002, ****P < 0.0001, ns = not significant.
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epidermal peels after P/MAMP treatment were significantly higher
than those produced in untreated epidermal peels of A. thaliana.
These data are similar to the reported results of triggered ethylene
biosynthesis in Col-0 plants after flg22 or nlp20 exposure.[26b]
Figure 6. Leaves of A. thaliana imaging of ethylene accumulation in response
to P/MAMP (flg22 or nlp20). a) Schematic overview of the general pathway that
leads to PTI/MTI response. b) Fluorescence and brightfield images of ethylene
detection in A. thaliana plants subjected to a 12 h P/MAMP (flg22 or nlp20)
treatment. Scale bars, 100 µm. c) Normalized mean fluorescence intensities of
A. thaliana plants with P/MAMP treatment. Plotted data are the average of three
independent experiments. Each experiment was repeated for three times. Mean
fluorescent intensity was measured from five individual leaf peels (fifteen
regions in random, 290.62 μm × 290.62 μm of each region) using ImageJ
software. Error bars denote ± SD. Statistical analysis was performed using one-
way ANOVA with Tukey’s multiple comparisons test. ***P < 0.0002, ****P <
0.0001. Error bars are ± SD (n = 15).
Figure 5. Detection of ethylene produced in A. thaliana mutants. a)
Fluorescence and brightfield imaging of epidermal peels applied with CEP-1 (10
µM) to the ethylene biosynthesis-defective acs2/6/4/5/9 and b) the phosphor-
mimicking 4myc-ACS6DDD. Scale bars, 100 µm. c) Quantification of the imaging
data (a and b). Plotted data are the average of three independent experiments.
Each experiment was repeated for three times. Mean fluorescent intensity was
measured from five individual leaf peels (fifteen regions in random, 290.62 μm
× 290.62 μm of each region) using ImageJ software. Error bars denote ± SD.
Statistical analysis was performed using one-way ANOVA with Tukey’s multiple
comparisons test. ****P < 0.0001. Error bars are ± SD (n = 15).
Conclusion
Plant innate immunity system triggered by perception of
Pathogen/Microbe associated molecular patterns (P/MAMPs) is
considered to be the first barrier to resistance the invasion of
pathogens or microbes. P/MAMPs can activate pattern-
recognition receptors (PRRs), which in turn initiate a variety of
downstream signalling events leading to the activation of a basal
resistance that is referred to P/MAMP-triggered immunity
(PTI/MTI). For instance, bacterial flagellin and NLPs (necrosis
and ethylene-inducing peptide 1 like proteins) are recognized by
pattern recognition receptors (PRRs) and to activate antimicrobial
defences. Which are usually linked with the ethylene biosynthesis,
accumulation of reactive oxygen species (ROS), cell wall cross-
linking and the release of secondary metabolites.[25] Depicted in
Figure 6a, flg22 is composed of a 22-amino acid sequence and
could induce plant defence mechanisms to produce more
ethylene; and nlp20 is a conserved 20-amino acid fragment that
could also result in immune activation to produce more ethylene
by cytotoxic and noncytotoxic NLPs.[26] As shown in Figure 6b, we
tested leaf epidermal peeled from old A. thaliana plants subjected
to local treatment with P/MAMP for plant ethylene accumulation.
As quantified in Figure 6c, the levels of ethylene produced in the
In summary, three fluorophore-tagged Rh(III)-based probes
were developed for the selective and quantitative detection of
ethylene. Quantitative analysis indicated that CEP-1 possessed
the highest sensitivity with a limit of detection of 52 ppb ethylene
in air. Moreover, CEP-1 exhibited high selectivity of ethylene and
avoided interference from other competing unsaturated
molecules of plant metabolites. Furthermore, we demonstrated
that CEP-1 has the potential to detect endogenous ethylene gas
not only in living CHO-K1 cells (in the presence of exogenously
added ethylene) but also endogenous ethylene produced in
tobacco and A. thaliana plants. These fluorophore-tagged Rh(III)-
based probes provide a novel reaction mechanism for the spatial
real-time monitoring of endogenous ethylene in cells and plant
leaves. We expect that CEP-1 could serve as an effective tool for
better understanding of the regulation of ethylene biosynthesis
and ethylene signal transduction in living biological systems and
thus promote the ethylene-related physiological research in plants.
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RESEARCH ARTICLE
[19] a) S. Fernández-Alonso, T. Corrales, J. L. Pablos, F. Catalina, Sens.
Actuators, B 2018, 270, 256-262; b) Z. Li, Y. Q. Xu, H. L. Zhu, Y. Qian, Chem.
Sci. 2017, 8, 5616-5621.
Acknowledgements
The authors would like to thank Prof. Xiaorong Tao and Prof.
Suomeng Dong of Nanjing Agricultural University for helpful
discussions and insightful comments on the manuscript. This
work was financially supported by the National Key Research and
Development Program of China (2017YFD0200500 and
2018YFD0200100), the National Natural Science Foundation of
China (32072443) and Natural Science Foundation of Jiangsu
Province (BK20201323).
[20] S. P. Burg, E. A. Burg, Science 1965, 148, 1190-1196.
[21] D. O. Adams, S. F. Yang, Proc. Natl. Acad. Sci. U. S. A. 1979, 76, 170-174.
[22] S. Li, X. F. Han, L. Y. Yang, X. X. Deng, H. J. Wu, M. M. Zhang, Y. D. Liu, S.
Q. Zhang, J. Xu, Plant, Cell Environ. 2018, 41, 134-147.
[23] S. Joo, Y. D. Liu, A. Lueth, S. Q. Zhang, Plant J. 2008, 54, 129-140.
[24] Y. Fichman, G. Miller, R. Mittler, Mol. Plant 2019, 12, 1203-1210.
[25] a) C. M. J. Pieterse, A. Leon-Reyes, S. Van der Ent, S. C. M. Van Wees, Nat.
Chem. Biol. 2009, 5, 308-316; b) X. Du, S. Wang, F. Gao, L. S. Zhang, J.
H. Zhao, H. S. Guo, C. L. Hua, Sci. China: Life Sci. 2017, 60, 852-860;
c) I. Albert, L. S. Zhang, H. Bemm, T. Nürnberger, Mol. Plant-Microbe Interact.
2019, 32, 1038-1046; d) F. Zhou, A. Emonet, V. Denervaud Tendon, P.
Marhavy, D. S. Wu, T. Lahaye, N. Geldner, Cell 2020, 180, 440-453 e418.
[26] a) S. Oome, T. M. Raaymakers, A. Cabral, S. Samwel, H. Böhm, I. Albert, T.
Nürnberger, G. Van den Ackerveken, Proc. Natl. Acad. Sci. U. S. A. 2014,
111, 16955-16960; b) I. Albert, H. Böhm, M. Albert, C. E. Feiler, J. Imkampe,
N. Wallmeroth, C. Brancato, T. M. Raaymakers, S. Oome, H. Q. Zhang, E.
Krol, C. Grefen, A. A. Gust, J. J. Chai, R. Hedrich, G. Van den Ackerveken, T.
Nürnberger, Nat. Plants 2015, 1, 15140.
Conflict of interest
The authors declare no conflict of interest.
Keywords: bioimaging • hydrocarbon activations • fluorescent
probes • ethylene
[1] a) M. E. Saltveit, Postharvest Biol. Technol. 1999, 15, 279-292; b) T. T. Storch,
T. Finatto, M. Bruneau, M. Orsel-Baldwin, J. P. Renou, C. V. Rombaldi, V.
Quecini, F. Laurens, C. L. Girardi, J. Agric. Food Chem. 2017, 65, 7813-7826;
c) J. H. Zhang, D. Cheng, B. B. Wang, I. Khan, Y. H. Ni, J. Agric. Food Chem.
2017, 65, 7308-7319; d) Y. D. Liu, M. F. Tang, M. C. Liu, D. D. Su, J. Chen,
Y. S. Gao, M. Bouzayen, Z. G. Li, Small Methods 2020.
[2]
[3]
[4]
S. F. Yang, N.E. Hoffman, Annu. Rev. Plant Physiol. 1984, 35, 155-189.
B. M. Binder, J. Biol. Chem. 2020, 295, 7710-7725.
M. Santiago Cintrón, O. Green, J. N. Burstyn, Inorg. Chem. 2012, 51, 2737-
2746.
[5]
[6]
H. Pham-Tuan, J. Vercammen, C. Devos, P. Sandra, J. Chromatogr. A 2000,
868, 249-259.
a) F. J. M. Harren, J. Mandon, S. M. Cristescu, in Encyclopedia of Analytical
Chemistry, 2012; b) S. M. Cristescu, J. Mandon, D. Arslanov, J. De Pessemier,
C. Hermans, F. J. M. Harren, Ann. Bot. 2013, 111, 347-360.
a) M. A. G. Zevenbergen, D. Wouters, V. A. T. Dam, S. H. Brongersma, M.
Crego-Calama, Anal. Chem. 2011, 83, 6300-6307; b) B. Esser, J. M. Schnorr,
T. M. Swager, Angew. Chem., Int. Ed. 2012, 51, 5752-5756.
O. Green, N.A. Smith, A.B. Ellis, J. N. Burstyn, J. Am. Chem. Soc. 2004, 126,
5952-5953.
[7]
[8]
[9]
B. Esser, T. M. Swager, Angew. Chem., Int. Ed. Engl. 2010, 49, 8872-8875.
[10] Y. Hitomi, T. Nagai, M. Kodera, Chem. Commun. (Camb) 2012, 48, 10392-
10394.
[11] a) F. H. Hu, Z. X. Chen, L. Y. Zhang, Y. H. Shen, L. Wei, W. Min, Angew. Chem.,
Int. Ed. 2015, 54, 9821-9825. b) S. Benson, A. Fernandez, N. D. Barth, F. de
Moliner, M. H. Horrocks, C. S. Herrington, J. L. Abad, A. Delgado, L. Kelly, Z.
Y. Chang, Y. Feng, M. Nishiura, Y. Hori, K. Kikuchi, M. Vendrell, Angew. Chem.,
Int. Ed. 2019, 58, 6911-6915. c) F. de Moliner, K. Knox, D. Gordon, M. Lee, W.
J. Tipping,A. Geddis,A. Reinders, J. M. Ward, K. Oparka, M. Vendrell, Angew.
Chem., Int. Ed. 2021, 60, 7637-7642.
[12] a) D. A. Iovan, S. Jia, C. J. Chang, Inorg. Chem. 2019, 58, 13546-13560. b)
K. J. Bruemmer, S. W. M. Crossley, C. J. Chang, Angew. Chem., Int. Ed. 2020,
59, 13734-13762.
[13] S. N. W. Toussaint, R. T. Calkins, S. Lee, B. W. Michel, J. Am. Chem. Soc.
2018, 140, 13151-13155.
[14] M. T. Sun, X. Yang, Y. N. Zhang, S. H. Wang, M. W. Wong, R. Y. Ni, D. J.
Huang, J. Agric. Food Chem. 2019, 67, 507-513.
[15] K. Vong, S. Eda, Y. Kadota, I. Nasibullin, T. Wakatake, S. Yokoshima, K.
Shirasu, K. Tanaka, Nat. Commun. 2019, 10, 5746.
[16] P. P. Duan, X. Lan, Y. Chen, S. S. Qian, J. J. Li, L. Lu, Y. B. Lu, B. Chen, M.
Hong, J. Zhao, Chem. Commun. (Camb) 2014, 50, 12135-12138.
[17] Y. Chen, D. Q. Wang, P. P. Duan, R. Ben, L. Dai, X. R. Shao, M. Hong, J.
Zhao, Y. Huang, Nat. Commun. 2014, 5, 4610.
[18] Q. Wu, Y. Chen, D. Y. Yan, M. Y. Zhang, Y. Lu, W. Y. Sun, J. Zhao, Chem.
Sci. 2017, 8, 169-173.
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Entry for the Table of Contents
Fluorophore-tagged Rh(III)-based fluorogenic probes (see picture) were developed for rapid (< 2 min), highly selective, and quantitative
detection of ethylene. Additionally, we demonstrated that CEP-1 has the capacity to detect endogenous ethylene gas in living cells and
plants.
9
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