plants

Article
In Silico Conformation of the Drug Colchicine into Tubulin Models
and Acute Phytotoxic Activity on Cucumis sativus Radicles
Omar Aristeo Peña-Morán 1, * , Jesús Jiménez-Pérez 2 , Litzia Cerón-Romero 2 and Maribel Rodríguez-Aguilar 1

1 División de Ciencias de la Salud, Universidad Autónoma del Estado de Quintana Roo,

Chetumal 77039, Quintana Roo, Mexico; [email protected]
2 División Académica de Ciencias Básicas, Universidad Juárez Autónoma de Tabasco,
Cunduacán 86690, Tabasco, Mexico; [email protected] (J.J.-P.); [email protected] (L.C.-R.)
* Correspondence: [email protected]

Abstract: Many tests are used to determine the toxic activity of miscellaneous substances, and those
that are simple, fast, and inexpensive are useful for screening compounds with applications in
different fields. The Cucumis sativus root growth inhibition test is an example of acute toxicity deter-
minations. On the other hand, colchicine has been used as a herbicide to generate polyploids in plant
species finally reaching the environment; for this reason, colchicine could become a point of attention
in ecotoxicology. This work established that Cucumis sativus, at the colchicine binding site (CBS)
in tubulin, shares 100% similarity with humans. Colchicine was docked on seven Cucumis sativus
computational models of the αβ-tubulin heterodimer, allowing us to understand a possible confor-
mation in tubulin to trigger its antimitotic effect. Furthermore, an in vitro phytotoxicity assay of
colchicine-treated cucumber radicles indicated a hormetic-type concentration-dependent response
with macroscopic changes in radicles and hypocotyl. These results support the highly preserved
Citation: Peña-Morán, O.A.; grade of tubulins in several species, and using microtubule inhibitors could require attention in
Jiménez-Pérez, J.; Cerón-Romero, L.; ecotoxicological issues. The Cucumis sativus root growth test could help evaluate small molecules
Rodríguez-Aguilar, M. In Silico (colchicine analogs), chiefly by CBS interactions, a known druggable site, still a target in the search
Conformation of the Drug Colchicine for antimitotic compounds.
into Tubulin Models and Acute
Phytotoxic Activity on Cucumis sativus Keywords: colchicine; Cucumis sativus; microtubules; phytotoxicity; tubulin-models
Radicles. Plants 2022, 11, 1805.
https://doi.org/10.3390/
plants11141805

Academic Editors: Urszula Krasuska 1. Introduction

and Katarzyna Ciacka In the ecotoxicology field, many pharmaceutical products pollute the environment
Received: 21 June 2022
once used and excreted, both for humans and animals. For decades, observations in na-
Accepted: 6 July 2022
ture and laboratory investigations have provided evidence of drugs in water, sediments,
Published: 8 July 2022
sludge, etc. [1], which leads to exposure of living organisms in the environment to phar-
maceutical products. However, much remains unknown about the effects of drugs on
Publisher’s Note: MDPI stays neutral
organisms in the ecosystem, particularly plants, and even more on the amounts of sub-
with regard to jurisdictional claims in
stances required to initiate a toxicological impact. Drug receptors in humans are, in most
published maps and institutional affil-
cases, highly conserved proteins between species, which may imply a similar mode of
iations.
action, and published studies have used molecular docking as a potential tool to predict
drug effects in aquatic organisms with exciting results [2].
Plant bioassays have become a valuable and efficient tool for monitoring toxicity in
Copyright: © 2022 by the authors.
laboratory investigations due to their sensitivity, simplicity, and quick results [3]. In addi-
Licensee MDPI, Basel, Switzerland. tion, these assays are characterized by the low cost of preparation and maintenance and the
This article is an open access article possibility of analyzing germination and growth parameters, such as germination rate, root
distributed under the terms and elongation, biomass, or enzymatic activity [4,5]. Tests that positively or negatively evaluate
conditions of the Creative Commons radicle growth and seed germination have been used to assess physical, chemical, and
Attribution (CC BY) license (https:// biological agents [6] to study concentration-response relationships under specific and con-
creativecommons.org/licenses/by/ trolled conditions [7–10]. For this purpose, multiple plant species have been used to deter-
4.0/). mine the phytotoxicity of physical, chemical, and biological agents, such as Cucumis sativus

Plants 2022, 11, 1805. https://doi.org/10.3390/plants11141805 https://www.mdpi.com/journal/plants

Plants 2022, 11, 1805 2 of 16

(cucumber) [11–14], Lactuca sativa (lettuce) [15], Raphanus sativus (radish) [16], Trifolium
pratense (red clover) [17], Medicago sativa (alfalfa) [18], Triticum aestivum (wheat) [17,19], etc.
The root growth inhibition test has been used to explore the toxicity of compounds
and extracts, and it has been used as an additional test to those carried out with the classic
toxicity methods since the results have demonstrated a correlation with other models. An
example of this was the 83% efficiency that the seed germination bioassay showed by
detecting 5 out of 6 antitumor compounds [6] and a 100% efficiency when associated with
the Artemia salina toxicity test with positive efficiency criteria when the mean Inhibitory
Concentration (IC50 ) is less than 10 µg/mL [5,20].
The root growth inhibition test is a set of metabolic and morphogenetic processes
in which plants pass through different phases until a photosynthetically active plant is
formed. It lasts from metabolic reactivation to seedling development (radicle, hypocotyl,
and cotyledons). Plant cells are divided by mitosis or meiosis, depending on the cell
line type (somatic or germline). Both processes are relatively similar in animal and plant
cells [21]. In the cell division process, chromatin condensation begins during the prophase,
and chromosomes become visible and are aligned precisely on the metaphase plate in the
middle of the mitotic spindle, which resembles a barrel shape because it lacks centrioles
that are not necessary for cell division [22], and its fibers are fascicles of microtubules. In
plants, cell division is determined by the phragmoplast, which consists of microtubules
formed during late anaphase or early telophase [23,24].
Tubulins are globular proteins that form the mitotic spindle; they are created by
heterodimers of α- and β-tubulins. Both tubulin sequences are very similar, with ap-
proximately 40% identity [25,26], and both are the main components of microtubules.
Eukaryotic cell microtubules are among the three main cytoskeleton components and
proteins with high sequence conservation in eukaryotic organisms [27]. They are highly
dynamic structures that continuously alternate between their assembly (straight active
structure) and disassembly (inactivate structure in curved form) [28]. The coexistence
of growing and shrinking microtubules under the same conditions is termed “dynamic
instability,” and their regulation is well understood [29]. The αβ-tubulin heterodimers
target many molecules known as microtubule interfering agents (MIA); some are used
in clinics for cancer treatment, and others in current investigations [30,31]. Furthermore,
three different binding sites of microtubules are known: the colchicine binding site (CBS)
(microtubule destabilizers), the vinca alkaloid binding site (microtubule destabilizers), and
the taxane binding site (microtubule stabilizers) [32]. Currently, research on ligands binding
the CBS on tubulin is ongoing.
Colchicine (COL) is a naturally occurring active alkaloid from Colchicum autumnale
(Figure 1). In the 1940s, its powerful antitumor effect was described; however, it was
withdrawn from clinical studies due to its high cytotoxic activity [33]. It is currently used
to treat gout and other inflammatory diseases [34–36]. COL is also a herbicide; it binds to
tubulin dimers and inhibits microtubule formation. Loss of spindle microtubules affects
nuclear division and chromosome separation. The lack of cortical microtubules interrupted
cell and tissue morphogenesis [37]. COL binds reversibly and selectively to microtubules,
particularly in the polar union of α- and β-tubulin, disturbing the mitotic spindle and
causing microtubule depolymerization in eukaryotic cells. The compound has also been
used for research in cytogenetics as an antimitotic due to its inhibitory effect on cell division
in plants and animals, either allowing chromosomes to be observed or exploring several
cell replications processes at the metaphase or anaphase [38].
Cucumis sativus is an annual herbaceous plant with a robust root system; seeds are
approximately 0.8–1.0 cm long and 0.3–0.5 cm wide. The germination time is three to
four days after sowing [39]. Cucumber seeds are easy to obtain due to their agricultural
importance, have been previously used to evaluate the toxicity of several substances, and
have been established as a COL-sensitive species [11–14,40]. However, the molecular
toxicology that triggers the antimitotic effect of COL in the radicles of C. sativus has not
been explained.
Plants 2022, 11, x FOR PEER REVIEW
Plants 2022, 11, 1805 3 of 16

Figure Chemical
Figure1.1. structure
Chemical of the of
structure drug
thecolchicine.
drug colchicine.
This work aimed to explain the binding mechanism of colchicine on Cucumis sativus
Cucumis
radicles sativus
and models by inisvitro
an annual herbaceous
and in silico plant
approaches. Thewith a robust
importance root
of this system; se
study
approximately
lies 0.8–1.0
in explaining the cm long
mechanism and of
of action 0.3–0.5
COL incm wide. The models
computational germination
of the α-time
and is three
β-tubulin heterodimer of Cucumis sativus, informing how similar the
days after sowing [39]. Cucumber seeds are easy to obtain due to their agricultdimer is compared
with its homologues in humans since this could represent a point of attention for the
portance, have been previously used to evaluate the toxicity of several substan
environmental exposure of the drug COL and other inhibitors of tubulins in the CBS, as
haveasbeen
well established
the potential as a risk
ecological COL-sensitive
when COL is species
used as a[11–14,40]. However, the
herbicide. Furthermore, we molecu
icology
used thatgrowth
the root triggers
test the antimitotic
as a low-cost, effect of COL
straightforward in the
strategy radicles
for plant of and
extracts C. sativus
pure has n
explained.
compound evaluations to detect those with cytotoxic potential, and this test could evaluate
other types
This of molecules
work aimed in to
different
explainfields,
theincluding
bindingthe pharmaceutical
mechanism field.
of colchicine on Cucumi
radicles
2. Results and models by in vitro and in silico approaches. The importance of this st
and Discussion
in explaining
2.1. theAlignment
Protein Sequence mechanism of action
Comparison of COLofin
and Modeling thecomputational models
αβ-Tubulin Heterodimers of of the α
C. sativus heterodimer of Cucumis sativus, informing how similar the dimer is co
tubulin
withFive
its α-tubulin
homologues chain in sequences
humans from cucumber
since aligned
this could showed a ahigh
represent identity,
point and
of attention for
the average identity percentage (I%) was 90.2% ± 3.6%. Seven β-tubulin sequences from
vironmental exposure of the drug COL and other inhibitors of tubulins in the CBS
cucumber-aligned chains indicated an I% of 92.3% ± 1.5%. In both comparisons, a high I%
as the
was potential
shared ecological
by the UniProt risk
database when COL
sequences is used as
(the alignments canabeherbicide.
observed inFurthermore,
detail in w
the root growth
Supplementary test as
Materials a low-cost,
in Tables straightforward strategy for plant extracts a
S1 and S2).
compound evaluations to detect those withchains
The comparisons between the α- and β-tubulin from cucumber
cytotoxic potential, against human
and this test coul
chains also showed high I% and similarity percentage (S%) (percentages and standard
ate other types of molecules in different fields, including the pharmaceutical field
deviation can be observed in Table 1).
A high degree of evolutionary conservation of chains was found, which may be due to
2. Results
the animal and and Discussion
vegetable tubulins derived from a common ancestor [24,27]. Those results
suggest a high degree of
2.1. Protein Sequence Alignmentconservation between the
Comparison primary
and protein
Modeling sequences
of the of both
αβ-Tubulin Heterodim
cucumber and human proteins.
C. sativus
The sequence of α-tubulin A0A0A0K6A8 showed the highest I% (84.7%, Table 1)
and wasFive α-tubulin
applied for the chain
α-tubulinsequences from cucumber
chain modeling. aligned
All subsequent modelsshowed a high ident
of αβ-tubulin
heterodimers were built with the A0A0A0K6A8 sequence because with
the average identity percentage (I%) was 90.2% ± 3.6%. Seven β-tubulin sequenc the COL-interacting
residues at the CBS in the human sequence (αS178, αT179, αA180, and αV181), only the
cucumber-aligned chains indicated an I% of 92.3% ± 1.5%. In both comparisons, a
αA180 residue was changed by the physiochemically similar residue αS180 for all five
was shared
cucumber by the UniProt database sequences (the alignments can be observed i
sequences.
in Supplementary MaterialsatintheTables
The alignment of sequences S1 and
CBS (only S2).
for the β-tubulin chain) was performed
with the residues
The interactingbetween
comparisons with colchicine at and
the α- a 4 Å β-tubulin
distance. Allchains
seven β-tubulin cucumber against
from cucumber
sequences were aligned with the human β-tubulin sequence
chains also showed high I% and similarity percentage (S%) (percentages (UniProt: Q9BVA1). The and s
I% (70.6%) and S% (100%) were obtained for all comparisons. The alignments of residues
deviation
in the CBS cancanbebe observed
observed in Table
in Table 2. 1).
A high degree of evolutionary conservation of chains was found, which may
to the animal and vegetable tubulins derived from a common ancestor [24,27]. T
sults suggest a high degree of conservation between the primary protein sequences
cucumber and human proteins.
The sequence of α-tubulin A0A0A0K6A8 showed the highest I% (84.7%, Tabl
was applied for the α-tubulin chain modeling. All subsequent models of αβ-tubu
Plants 2022, 11, 1805 4 of 16

Table 1. Alignment of complete α- and β-tubulin sequences of C. sativus against H. sapiens (P68363
for α-tubulin chain and Q9BVA1 for β-tubulin chain).

Entry Length Identity (%) Similarity (%)

α-tubulin
A0A0A0K6A8 450 84.7 97.6
A0A0A0LFM5 449 81.8 96.7
A0A0A0KWR8 449 81.4 96.7
A0A0A0KWB5 450 84.0 98.0
A0A0A0KIM4 415 77.8 97.6
Mean 442.6 ± 15.4 82.0 ± 2.7 97.3 ± 0.6
β-Tubulin
A0A0A0L2I9 446 84.5 95.7
A0A0A0LTS3 445 83.6 96.4
A0A0A0LCY8 446 83.0 95.5
A0A0A0LPG6 449 83.3 96.4
A0A0A0LXT7 447 83.4 94.6
A0A0A0LVT8 443 86.1 96.6
A0A0A0KQW7 441 82.9 97.1
Mean 445.3 ± 2.6 83.8 ± 1.1 96.1 ± 0.8

Table 2. In situ protein sequence alignment between the residues forming the CBS, Z-score, and
percentage of residues in the Ramachandran plot analysis within favored and allowed regions.

β-Tubulin Residues in the CBS

Uniprot ID Model Z-Score RP 1 (%)
238 241 242 248 250 251 254 255 258 259 314 315 316 318 350 352 378

Q9BVA1 V C L L A D K L N M T V A I N K I - - -
V C L L S D K L N L T A S M N K I
A0A0A0L2I9 m1 −9.36 96.2
* * * * : * * * * : * . : : * * *
V C L L S D K L N L T A S M N K I
A0A0A0LTS3 m2 −10.01 96.4
* * * * : * * * * : * . : : * * *
V C L L S D K L N L T A S M N K I
A0A0A0LCY8 m3 −9.65 96.4
* * * * : * * * * : * . : : * * *
V C L L S D K L N L T A S M N K I
A0A0A0LPG6 m4 −9.95 96.9
* * * * : * * * * : * . : : * * *
V C L L S D K L N L T A S M N K I
A0A0A0LXT7 m5 −9.39 96.6
* * * * : * * * * : * . : : * * *
V C L L S D K L N L T A S M N K I
A0A0A0LVT8 m6 −9.48 96.6
* * * * : * * * * : * . : : * * *
V C L L S D K L N L T A S L N K I
A0A0A0KQW7 m7 −9.81 97.5
* * * * : * * * * : * . : : * * *

1 Ramachandran Plot. (*) Same residue. (:) Conservatives residues. (.) Semi-conservative residue.

The TASSER models of tubulin macromolecules and the validation method results
allowed us to know the overall quality of the models (the one α-tubulin model and the
seven β-tubulin model) through Z-score calculations. The typical Z-score value for proteins
around 445 residues lies between −3.9 and −13.0. Table 2 shows the Z-score for each
β-tubulin model within the typical Z-score interval. The Z-score graphs of all models are
detailed in Table S3, available in the Supplementary Materials. On the other hand, the
Ramachandran plots of all models showed residues percentages in the favored and allowed
regions of 96.2–97.5% limits. In addition, it was manually verified that none of the residues
forming the CBS were in non-allowed regions. The Ramachandran plots for each model
are available as Supplementary Materials (Table S3).
Comparative analysis of the β-tubulin chains between the human (Q9BVA1) and
cucumber sequences revealed five changed residues: A250, M259, V315, A316, and I318
to S248, L257, A313, S314, and M316, respectively. The β-tubulin sequence fragment
corresponding to the A0A0A0KQW7 UniProt entry replaced M318 (found in the rest of the
sequences) with L316 (Table 2). For all seven C. sativus compared sequences, four sites with
conservative (:) and one with semi-conservative (.) replacements were found.
The comparative results suggest that the CBS in all analyzed cucumber sequences was
preserved compared to human sequences, particularly in the gene TUBB2B, which can be
detected in many human tissues, mainly in the brain and male tissue [41]. These results
 with conservative (:) and one with semi-conservative (.) replacements were found.
The comparative results suggest that the CBS in all analyzed cucumber sequences
was preserved compared to human sequences, particularly in the gene TUBB2B, which
can be detected in many human tissues, mainly in the brain and male tissue [41]. These
Plants 2022, 11, 1805could clarify why COL is used as a phytotoxic compound on many plant species. 5 of 16
results
Proteins in the tubulin family have a high preservation degree, and minimal changes in
the tubulin sequence have probably been imposed by the structural limitations of the as-
could clarify why COL is used as a phytotoxic compound on many plant species. Proteins
sembly and disassembly of microtubules
in the tubulin family have and
a highthe constraints
preservation imposed
degree, by thechanges
and minimal tubulin in fam-
the tubulin
ily association proteins such as kinesins and dyneins [27].
sequence have probably been imposed by the structural limitations of the assembly and
The crystallized structure
disassembly of of tubulin was
microtubules and theobtained from
constraints the by
imposed PDB database
the tubulin (4O2B,
family association
proteins such as kinesins and dyneins [27].
X- ray diffraction method, resolution: 2.3 Å). The crystallized model includes two hetero-
The crystallized structure of tubulin was obtained from the PDB database (4O2B, X-ray
dimers of α- and β-tubulin, a stathmin molecule, and tubulin–tyrosine ligase chains. In
diffraction method, resolution: 2.3 Å). The crystallized model includes two heterodimers
addition, because COL of α-was co-crystallized,
and β-tubulin, it was
a stathmin possible
molecule, to situate the CBS
and tubulin-tyrosine and
ligase compare
chains. In addition,
its conformation [42,43]. because COL was co-crystallized, it was possible to situate the CBS and compare its
The TASSER modelsconformation
with[42,43].
the highest C-score were selected, typically in (−5, 2),
The TASSER models with the highest C-score were selected, typically in (−5, 2), where
where a higher C-score value means a model with increased confidence and vice-versa
a higher C-score value means a model with increased confidence and vice-versa [44–46].
[44–46]. An image of Anthe seven
image aligned
of the monomers
seven aligned of β-tubulin
monomers andand
of β-tubulin a close-up
a close-upview
viewatatthe
the CBS
CBS showing the conformation adopted by the COL can be observed in Figure
showing the conformation adopted by the COL can be observed in Figure 2. 2.

Figure 2. All seven TASSER αβ-tubulin

Figure 2. All seven TASSERmodels of C.models
αβ-tubulin sativus
of (each in(each
C. sativus different colors)
in different aligned
colors) alignedto
to 4O2B
4O2B macromolecule,macromolecule,
and a close-up view of the CBS with co-crystallized COL (black sticks).
and a close-up view of the CBS with co-crystallized COL4O2B (black sticks). Note that
4O2B

Note that all models fit

allthe tertiary
models fit thestructure of theof4O2B
tertiary structure model,
the 4O2B model,andandqualitatively, the
qualitatively, the CBSCBS is highly
is highly conserved.
conserved.
The CBS (named RB3-SLD by Ravelli et al., in 2004 [28]) is mainly found in the inter-
mediate domain of the β-tubulin, placed between the S8 and S9 strands, the T7 turn, and
The CBS (namedthe RB3-SLD by H8
helices H7 and Ravelli et2).al.,
(Figure COLin also
2004interacts
[28]) iswith
mainly
the T5found
loop ofin
thethe inter-chain
α-tubulin
mediate domain of mainly
the β-tubulin, placed between the S8 and S9 strands, the T7 turn, and
by αT179 electrostatic interactions, and this interaction stabilizes the αβ-tubulin
the helices H7 and heterodimer
H8 (Figurepreventing
2). COLtubulin
also interacts
from ongoingwith thea curved
from T5 loopto aof the α-tubulin
straight conformation.
chain mainly by αT179 electrostatic interactions, and this interaction stabilizes the Homo
The results of the sequence comparison between Cucumis sativus and αβ-tu-sapiens
tubulins have suggested that proteins are presenting conserved regions, but also changed
bulin heterodimer preventing tubulin from ongoing from a curved to a straight confor-
residues have been found in the CBS; however, these residues are semi-conservative or
mation. conservative, which could lead COL to obtain a stable conformation at the same mode
already reported. In addition, the modeling and alignment originated seven heterodimers
of αβ-tubulin from the protein sequences of C. sativus. Finally, an in silico molecular
docking analysis was performed to understand how the COL could interact with the CBS.

2.2. In Silico Docking Studies between Colchicine and the C. sativus αβ-Tubulin Models
The in silico molecular docking validation analysis with COL (COLDocked ) showed
a cluster of lowest energy of 53/7 (poses in the lowest energy cluster/total number of
clusters). The pose with the lowest energy at the cluster, the mean energy in the cluster, Ki,
and RMSD computed by comparison with co-crystallized COL4O2B , are shown in Figure 3.
 2.2. In Silico Docking Studies between Colchicine and the C. sativus αβ-Tubulin Models
The in silico molecular docking validation analysis with COL (COLDocked) sho
cluster of lowest energy of 53/7 (poses in the lowest energy cluster/total number o
ters). The pose with the lowest energy at the cluster, the mean energy in the clus
Plants 2022, 11, 1805 6 of 16
and RMSD computed by comparison with co-crystallized COL4O2B, are shown in Fi

Figure 3. Molecular docking validation. Image of ligand poses between the co-crystallized ligand
Figure 3. ,Molecular
(COL4O2B docking
green) [47] and validation.
the mean-energy poseImage of ligand
found with poses
the docking between
algorithm the
(COL co-crystallized
Docked , red)
(COL 4O2B, green) [47] and the mean-energy pose found with the docking algorithm (COLDoc
are shown.
are shown.
The atomic details of the CBS were revealed in 2004 when Ravelli et al., published the
structure of tubulin with the stathmin-type domain of the RB3 protein in the colchicine
The atomic details of the CBS were revealed in 2004 when Ravelli et al., pub
complex (PDB: 1SA0); however, higher resolution structures were obtained with tubulin–
the
tyrosine ligaseof[47].
structure tubulin with the stathmin-type
Superimposition domainconformation
between the colchicine of the RB3 protein in the colc
previously
complex
reported by (PDB:
Prota1SA0); however,
et al. in 2014 and thehigher resolution
conformation obtainedstructures
by dockingwere obtained
analysis with the with tu
C. sativus model was performed. It can be visualized in Figure
tyrosine ligase [47]. Superimposition between the colchicine conformation 3. The lowest mean energypreviou
conformer of COLDocked was found like that already reported in the PDB as 4O2B crystal
ported by Prota et al. in 2014 and the conformation obtained by docking analysis w
by Prota et al. in 2014. Once the RMSD at the validation analysis was <2 Å, the docking
C.parameters
sativus model was performed.
were acceptable for furtherItinvestigations.
can be visualized in Figure 3. The lowest mean e
conformer of COLdocking
The molecular Docked was foundoflike
analysis the that already
CBS with reported
the COL ligandinand
theall
PDB as 4O2B
seven
αβ-tubulin models from C. sativus showed 8 to 14 conformational
by Prota et al. in 2014. Once the RMSD at the validation analysis was <2 Å, the d clusters after 100 runs.
For those with
parameters the acceptable
were lowest conformational
for further energy, the mean free Gibbs energy (∆Gb in
investigations.
Kcal/mol), the inhibition constant (Ki) obtained from the standard Gibbs free energy equa-
tion,The molecular
and the docking
RMSD computed foranalysis
each pose of thecluster
in the CBS by with the COLcomparison
the COL 4O2B
ligand and are all sev
tubulin
reportedmodels from
in Table 3. C. sativus
For the models m5, showed
m6, and 8 to
m7,14 conformational
a low number of poses clusters after 100 ru
was obtained
those
in the with
lowest the lowest
energy cluster.conformational
The rest of the models energy, the between
obtained mean free 26–41Gibbs energy (Δ
poses. The
energy interval obtained for COL in all C. sativus models was about −
Kcal/mol), the inhibition constant (Ki) obtained from the standard Gibbs free energy8.88 ± 0.58 Kcal/mol
with a percentage coefficient of variation of 6.54%.
tion, and the RMSD computed for each pose in the cluster by the COL4O2B comparis
reported in Table
Table 3. Results from3.
theFor the models
molecular dockingm5, m6,into
analysis andthem7,
CBSabetween
low number
COL andof
theposes
seven was ob
inαβ-tubulin
the lowest energy
heterodimer cluster.
models The rest of the models obtained between 26–41 pose
of C. sativus.
energy interval obtained for COL in all C. sativus models was about −8.88 ± 0.58 Kc
Model Poses/Clusters 1 ∆Gb (Kcal/mol) Ki (µM) RMSD
with a percentage coefficient of variation of 6.54%.
m1 28/8 −8.99 ± 0.59 0.412 1.12 ± 0.39
m2 26/10 −8.79 ± 0.45 0.606 1.11 ± 0.31
Table 3.m3
Results from the molecular docking
30/9 analysis into0.145
−9.56 ± 0.51 the CBS between
1.01 ±COL
0.35 and the sev
m4 41/8 − 9.65
tubulin heterodimer models of C. sativus. ± 0.21 0.091 1.18 ± 0.79
m5 2/10 −8.44 ± 1.01 1.195 1.02 ± 0.57
m6 2/14 −18.72 ± 1.02 0.744 1.01 ± 0.51
Model Poses/Clusters ΔGb (Kcal/mol) Ki (µM) RMSD
m7 4/10 −8.03 ± 0.67 1.988 1.58 ± 0.26
1 Poses inm1 28/8number of clusters.
the lowest energy cluster/total −8.99
± 0.59 0.412 1.12 ± 0
m2 26/10 −8.79 ± 0.45 0.606 1.11 ± 0
Differences in primary sequence can be found in each model of the β-tubulin proteins
from C. sativus (Table S2). However, the COL affinity of binding into the CBS is not
affected. In addition, the in silico model minimization did not affect the pocket of binding.
Furthermore, all conformations in the lowest energy cluster are like that adopted by COL
in the 4O2B crystal (RMSD < 2 Å). As previously reported, those results suggest that COL
could bind to CBS in C. sativus tubulin and consequently giving a phytotoxicity induction
effect and antimitotic activity [11–14,40].
 Differences in primary sequence can be found in each model of the β-tubulin proteins
from C. sativus (Table S2). However, the COL affinity of binding into the CBS is not af-
fected. In addition, the in silico model minimization did not affect the pocket of binding.
Furthermore, all conformations in the lowest energy cluster are like that adopted by COL
Plants 2022, 11, 1805
in the 4O2B crystal (RMSD < 2 Å). As previously reported, those results suggest that 7COL
of 16
could bind to CBS in C. sativus tubulin and consequently giving a phytotoxicity induction
effect and antimitotic activity [11–14,40].
Figure 4 compares the lowest energy conformation adopted by the COL of all the
Figure 4 compares the lowest energy conformation adopted by the COL of all the
models used in this study. The complete interaction network between COL and the mod-
models used in this study. The complete interaction network between COL and the models
els m1–m7 can be observed in Table S4. In addition, the 4 Å interacting residues of αβ-
m1–m7 can be observed in Table S4. In addition, the 4 Å interacting residues of αβ-tubulin
tubulin are shown in Table S4.
are shown in Table S4.

Figure 4. All COL poses merged. The figure shows the lowest-energy conformations achieved
Figure 4. All COL poses merged. The figure shows the lowest-energy conformations achieved from
from molecular docking of COL with the αβ-tubulin models from C. sativus. The conformations
molecular docking of COL with the αβ-tubulin models from C. sativus. The conformations showed
showed
that that the orientation
the orientation of the functional
of the functional groups isgroups is maintained
maintained in the
in the CBS CBS compared
compared with thewith the
control
control (4O2B).
(4O2B).

The results
The results in
in the
the in
in silico
silico molecular
molecular docking
docking between
between COL
COL and
and CBS
CBS in
in the
the cucumber
cucumber
model suggest a strongly related conformation in the 4O2B model. Furthermore, the
model suggest a strongly related conformation in the 4O2B model. Furthermore, the align-
alignment between COL conformations obtained in this work (Figure 4) and that found in
ment between COL conformations obtained in this work (Figure 4) and that found in the
the crystallized structure 4O2B also suggests that changes in the residues between cucumber
crystallized structure 4O2B also suggests that changes in the residues between cucumber
and human chains do not affect the binding affinity due to the conserved physicochemical
and human chains do not affect the binding affinity due to the conserved physicochemical
environment of amino acids in C. sativus sequences (conservative and semi-conservative).
environment of amino acids in C. sativus sequences (conservative and semi-conservative).
All complete interaction maps can be seen in Table S4 in the Supplementary Materials.
All complete interaction maps can be seen in Table S4 in the Supplementary Materials.
Finally, the root growth inhibition test was performed with the germinated radicles of
Finally, the root growth inhibition test was performed with the germinated radicles of
C. sativus to explore an in vitro concentration-dependent activity by COL.
C. sativus to explore an in vitro concentration-dependent activity by COL.
2.3. Technical Considerations before Starting the Phytotoxic Test
2.3. Technical Considerations before Starting the Phytotoxic Test
The viability of cucumber seeds was calculated to be 96% (n = 50), showing a high
Plants 2022, 11, x FOR PEER REVIEW seed The viability
viability rate of
in cucumber seeds was the
the lot. Additionally, calculated to be
treatment time96% (n for
(TT) = 50),
theshowing a high
COL treatment
8 of 16
seed viability rate in the lot. Additionally, the treatment time (TT) for the COL
was 3.56 days (85 h) after sowing. All assessed parameters can be observed in Figure 5. Atreatment
was
high3.56 days (85 h)
germination after sowing.
percentage was All assessed
crucial parameters
for the can be
phytotoxicity testobserved
because in Figure
it is based5.on
A
high germination
root elongation andpercentage was crucial
not germination for the phytotoxicity test because it is based on
capacity.
root elongation and not germination capacity.

Parameters Days
FDG 2.5 ± 0.0
LDG 4.63 ± 0.53
T 4.48 ± 0.04
TSG 1.06 ± 0.26
TT 3.56 ± 0.26

Figure 5. Cucumber seed germination parameters were calculated according to a previous phytotox-
icity5.assay.
Figure The graph
Cucumber seed shows the germination
germination parameterspercentage (%G) of
were calculated the seed to
according lotaof C. sativus
previous against
phyto-
germination
toxicity days,
assay. The lot viability,
graph shows the and calculated germination
germination factors.
percentage (%G) Each
of the point
seed lot in
of the graph against
C. sativus represents
the %G ± days,
germination standard deviationand
lot viability, (n =calculated
50) againstgermination
time (days).factors. Each point in the graph repre-
sents the %G ± standard deviation (n = 50) against time (days).

The first day of germination (FDG) indicates how fast the germination begins. For
the sown seeds of C. sativus, this time was determined in 2.5 days for the detection of the
first germinated seeds; for the LDG (last day of germination), the result indicated germi-
 Plants 2022, 11, 1805 8 of 16

The first day of germination (FDG) indicates how fast the germination begins. For the
sown seeds of C. sativus, this time was determined in 2.5 days for the detection of the first
germinated seeds; for the LDG (last day of germination), the result indicated germination
of 96% of the seeds at 4.63 h, which showed a difference of 2.1 days between the beginning
and the end of germination; the average germination time (T) indicated the time required
for viable seeds to germinate, which was found to be approximately 4.5 days (about 108 h);
the mean seed germination time (TSG) and FDG allowed us to detect the best treatment
time (TT) to start the trial with COL.

2.4. In Vitro Antimitotic Effect of Colchicine (Phytotoxicity)

Phytotoxicity assessment has enabled the potential use of COL as an inhibiting radicle
growth agent in cucumber seeds. The quantitative results in Figure 6 strongly suggest
that cucumber radicle growth was affected by treatment with COL in a concentration-
dependent approach; furthermore, the compound could induce hormesis after 48 h of
treatment. Thus, it has been established that hormesis is related to stress in living things,
Plants 2022, 11, x FOR PEER REVIEW the sum of non-specific biological responses to threatening stimuli and tends to disrupt
9 of 16

homeostasis. Hormesis is currently considered a non-specific adaptive response to low

doses of stressors [48].

Figure 6. In vitro antimitotic effect of colchicine on cucumber radicles. The graph indicates the aver-
Figure 6. In vitro antimitotic effect of colchicine on cucumber radicles. The graph indicates the av-
age percentage of primary root growth (%RG) for each concentration of COL on the Cucumis sativus
erage percentage of primary root growth (%RG) for each concentration of COL on the Cucumis sa-
radicles
tivus (n = (n
radicles 20)= at
20)24atand 48 h 48
24 and of hincubation. Concentrations
of incubation. Concentrationsof 2.5ofand
2.50.25
and mM
0.25 showed
mM showedsignificant
sig-
differences
nificant (* p < 0.05)
differences (* p <compared to the control
0.05) compared at 24 and
to the control 48and
at 24 h of48
treatment. The three
h of treatment. Thehighest concen-
three highest
trations were applied
concentrations for linear
were applied for regression analysis.
linear regression The IC50
analysis. Thewas
ICdetermined for eachfor
50 was determined quantified
each quan-day.
tified day. The regression
The regression coefficientscoefficients
of determination (R2 ) in the (R
of determination 2) in were
graph the graph wereclose
relatively relatively
to the close to the
unit and can
unit and can be
be observed forobserved for each analysis.
each analysis.

At a concentration
2.5. Macroscopic of 0.025
Characteristics mM, a stimulating
of Cucumber-Treated effect of radicle growth was observed;
Seeds
however, phytotoxic effects were observed at 0.25 mM
Macroscopic observations showed interesting results. afterThe
the morphology
first 24 h of incubation. The
of two repre-
COL-determined mean growth inhibitory concentration (IC
sentatives of germinated seeds was observed: control group (Figure 50 ) values
7, left) and post-treat-of
at 24 h and 48 h
treatment were 0.85 mM and 1.77 mM, respectively. In addition,
ment with 2.5 mM COL for 48 h (Figure 7, right). The COL treatment the results of Figure 6
morphological
showed
change that the minimum
suggested tolerable
an antimitotic effectconcentration
by decreasinginthe C. sativus
thegrowth radicles was
of cucumber 0.025 mM
radicles.
of COL since this concentration does not significantly affect the elongation of the radicles.
Radicle growth in cucumber germinated seeds could be used to screen for in vitro
antimitotic activity. A work limit concentration of 1 mg/mL (2.5 mM COL equivalence) is
recommended. COL has been used in phytotoxic concentration intervals from 0.25 mM to
38 mM to induce somatic polyploidies in vitro [49].
Although a large arsenal of compounds targeting microtubules is known [50,51], new
compounds targeting the CBS continue to be researched in depth due to the potential drug-
 Figure 6. In vitro antimitotic effect of colchicine on cucumber radicles. The graph indicates th
erage percentage of primary root growth (%RG) for each concentration of COL on the Cucum
Plants 2022, 11, 1805 tivus radicles (n = 20) at 24 and 48 h of incubation. Concentrations of 2.5 and 0.259 ofmM 16 showe
nificant differences (* p < 0.05) compared to the control at 24 and 48 h of treatment. The three hi
concentrations were applied for linear regression analysis. The IC50 was determined for each q
tified day.inThe
gable sites regressionrepresenting
microtubules, coefficients of determination
a possibility (R2) in the
for anticancer graph
drug were relatively
development [33]. close
unit and can be observed for each analysis.
However, the possible ecotoxicological impact that cytotoxic compounds could have on the
environment, such as those affecting the microtubules of cells, should be considered.
2.5. Macroscopic Characteristics of Cucumber-Treated Seeds
2.5. Macroscopic Characteristics of Cucumber-Treated Seeds
Macroscopic observations showed interesting results. The morphology of two r
Macroscopic observations showed interesting results. The morphology of two rep-
sentatives of germinated
resentatives of germinatedseeds
seeds was
was observed:
observed: control
control group
group (Figure
(Figure 7, left)7,and
left) and post-t
post-
ment with 2.5 mM COL for 48 h (Figure 7, right). The COL treatment
treatment with 2.5 mM COL for 48 h (Figure 7, right). The COL treatment morphological morpholo
change
change suggested
suggested an antimitotic
an antimitotic effect effect by decreasing
by decreasing theofgrowth
the growth cucumber ofradicles.
cucumber radicle

Figure
Figure 7. 7. Macroscopic
Macroscopic and microscopic
and microscopic analysisanalysis
of controlof control
and treatedand treatedseeds
germinated germinated seeds of cu
of cucumber.
ber.germinated
The The germinated control
control (seed on the(seed on the
left) shows the left)
typicalshows the typical
morphology morphology
of the primary of the primary
and secondary
secondary
roots, roots,
tegument, tegument,
cotyledon, cotyledon,
and hypocotyl canand hypocotyl
be observed. can be observed.
A morphological changeAcan
morphological
be observed chang
be observed in the germinated seeds treated with 2.5 mM of COL (seed on the right),
in the germinated seeds treated with 2.5 mM of COL (seed on the right), a broader and shortera broade
shorter hypocotyl
hypocotyl (gray arrow),(gray arrow),root
and primary and primary
blebbing root blebbing
formation formation
of approximately of (solid
1–2 mm approximately
circle), 1–2
and secondary
(solid circle),root
andinhibition
secondary (black
rootarrows).
inhibition (black arrows).

A blebbing formation can be observed at the division zone of the primary root in COL-
A blebbing formation can be observed at the division zone of the primary ro
treated radicles (Figure 7, right), and it is associated with the phytotoxic effect described
COL-treated
for radicles plants
COL. Dicotyledonous (Figure 7, aright),
have and
primary it that
root is associated with thetophytotoxic
repeatedly branches generate effec
scribed
lateral forthat
roots COL. Dicotyledonous
originate plants
exclusively from have afounder
pericycle primary rootThese
cells. that are
repeatedly
initiated branch
when individuals
generate lateralorroots
pairs of pericycle
that originatefounder cells undergo
exclusively fromseveral rounds
pericycle of anticlinal
founder cells. Thes
and periclinal divisions, followed by patterning and emergence, activation of the new
meristem, and lateral root elongation [52]. COL causes lateral root cell division inhibition
and decreased radicle growth on germinated cucumber seeds, previously reported as
polyploid formation in other plant species [53].
What is sought with the root growth test is a statistically significant decrease in the size
of the radicles of C. sativus and a concentration-dependent effect on the IC50 calculation;
those parameters make it possible to find the phytotoxic activity of COL. Additional
observations came from some morphological changes in the radicles by COL treatment.
As it was described in Figure 8, a blebbing formation can be observed at the division zone
of the primary root in COL-treated radicles, and it is associated with the antimitotic effect
described for COL that causes the inhibition of the lateral root division and decreases
radicle growth on germinated cucumber seeds, previously reported as polyploid formation
in other plant species. [53] Figure 8 shows the components in a primary root (Figure 8a),
and a C. sativus COL treated seed (Figure 8b). The comparison revealed that blebbing
formation could be found at the division zone in radicles, consistent with the effects of an
antimitotic compound when root growth is also decreased.
 of the primary root in COL-treated radicles, and it is associated with the antimitotic effect
described for COL that causes the inhibition of the lateral root division and decreases rad-
icle growth on germinated cucumber seeds, previously reported as polyploid formation
in other plant species. [53] Figure 8 shows the components in a primary root (Figure 8a),
and a C. sativus COL treated seed (Figure 8b). The comparison revealed that blebbing for-
Plants 2022, 11, 1805 10 of 16
mation could be found at the division zone in radicles, consistent with the effects of an
antimitotic compound when root growth is also decreased.

(a) (b)
Figure 8.Figure
Blebbing formation
8. Blebbing in the division
formation in the zone. (a) Indicates
division zone. (a) the segments
Indicates the and namesand
segments assigned
namestoassigned
a radicleto[23]. (b) The
a radicle figure
[23]. suggests
(b) The figureasuggests
C. sativus seed
a C. treated
sativus seedby COL 2.5
treated mM and
by COL the and
2.5 mM blebbing
the blebbing
formation in the division zone.
formation in the division zone.

The conservation of the significant

The conservation characteristics
of the significant of cell organization
characteristics and several
of cell organization and several
genes ingenes
eukaryotic organisms
in eukaryotic strengthens
organisms the theory
strengthens the that
theoryall that
existing eukaryotic
all existing forms forms
eukaryotic
have evolved from afrom
have evolved common
a commonancestor [54]. [54].
ancestor For example,
For example, plant cells
plant areare
cells derived
derivedfrom from undif-
undifferentiated
ferentiated cells called
cells calledmeristematic
meristematiccells,cells,and
andthrough
throughgermination
germinationororseedling
seedlingfor- formation,
mation,they
theyundergo
undergoconstant
constantcell
celldivision
divisiontotobecome
becomeaacomplete
completeplant.
plant.
Nowadays,Nowadays, it is unknown
it is unknown how COL howtreatments
COL treatments affect meristematic
affect meristematic cells in cells
roots.in roots.
However,However, the mechanism
the mechanism of action ofhas
action
been has been widely
widely studied;studied; it was
it was even even
more more challenging
challenging
to find crystallized
to find crystallized and elucidated
and elucidated structures
structures of the of the tubulin-COL
tubulin-COL complex complex
from from C. sativus in
C. sativus
the Protein Data Bank repository. However, the TASSER models
in the Protein Data Bank repository. However, the TASSER models have made it possible have made it possible to
to understand the conformation that COL could adopt between the α- and β-tubulin chains
understand the conformation that COL could adopt between the α- and β-tubulin
inthe
chains in C.sativus
theC. sativustubulin
tubulinmodels;
models;evenevenmore,
more,the theminimal
minimalchanges
changesofofresidues
residuesfound
found at the
sequenceswere
at the sequences wereconservative
conservativefor forthe
theCOL
COLdocking.
docking.According
Accordingtotothe theresults
resultsofofthis
this study,
theCOL
study, the COLmechanism
mechanism of of action
action between
between human
human and and plant
plant (cucumber)
(cucumber) models
models werewere similar,
similar,which
whichcouldcouldrepresent
representanan ecotoxicological
ecotoxicological risk depending
risk depending on the concentration
on the concentration that can be
found in the environment.
that can be found in the environment.
The authors
The authors considerconsider
that thethat
rootthe root growth
growth inhibitioninhibition assaybe
assay could could be a speedy
a speedy assay assay
to explore compounds with antimitotic effects at the CBS in tubulin: applying the testthe test
to explore compounds with antimitotic effects at the CBS in tubulin: applying
compounds
compounds at 1 mg/mLat 1 mg/mL
on a groupon a of
group of germinated
germinated seeds, seeds, then analyzing
then analyzing the morphological
the morphologi-
cal changes after the treatment (at least 24 or 48 h) and comparing them against aacontrol
changes after the treatment (at least 24 or 48 h) and comparing them against control group.
It is recommended to examine the viability of the seed lot and determine the FDG, LDG,
%G, T, TSG, and TT, to perform treatments with the most significant number of germinated
seeds. Advantages of this trial as a research strategy include low-cost, easy assembly, and
straightforward interpretations.

3. Materials and Methods

3.1. Protein Sequence Alignment Comparison and Modeling of the αβ-Tubulin Heterodimer of
C. sativus
A comparative bioinformatic analysis was performed with the α- and β-tubulin se-
quences between humans and cucumbers (Homo sapiens vs. Cucumis sativus). In the UniProt
database, five protein sequences corresponding to α-tubulin (UniProt: A0A0A0K6A8,
A0A0A0LFM5, A0A0A0KWR8, A0A0A0KWB5, and A0A0A0KIM4) and seven sequences
corresponding to β-tubulin (UniProt: A0A0A0L2I9, A0A0A0LTS3, A0A0A0LCY8,
A0A0A0LPG6, A0A0A0LXT7, A0A0A0LVT8, A0A0A0KQW7, and) were found, all se-
quences belonged to C. sativus. The α- and β-tubulin sequences from UniProt were aligned
with those from humans: α-tubulin chain 1B (UniProt: P68363) and β-tubulin chain 2B
(UniProt: Q9BVA1), respectively. The percentage of similarity (S%) and identity (I%) were
obtained by the UniProt Basic Local Alignment Search Tool (BLAST) [55]. In situ, the
Plants 2022, 11, 1805 11 of 16

residues forming the CBS [47] in both α- and β-tubulin were aligned for identity and
similarity determinations. The S% was calculated considering the radical-conservative
replacement ratios, volume, and polarity between residues [56]. Residues forming the
CBS (4 Å) were as follows: αS178, αT179, αA180, αV181, βC241, βL248, βA250, βD251,
βK254, βL255, βN258, βT314, βV315, βA316, βI318, βN350, and βK352.
One α-tubulin monomer of C. sativus was modeled using the α-tubulin sequence
A0A0A0K6A8 (due to its highest identity compared to the human α-tubulin protein);
likewise, all seven β-tubulin sequences from C. sativus found in the Uniprot database
were modeled. The TASSER server was used for this purpose [44–46]. In addition, the
PDB model of the α-tubulin chain from Homo sapiens (PDB: 5IJ0) [57] and the Bos taurus
β-tubulin chain co-crystallized with COL [43] (PDB: 4O2B; the β-tubulin chain in the 4O2B
model from the PDB database shares 445 identical residues (100% identity) with the human
β-tubulin chain in the UniProt alignment of Q9BVA1 vs. Q6B856) were used as templates
to guide the modeling.
The protein models of α- and β-tubulin chains of C. sativus were individually validated
with the Z-score, indicating overall model quality. The ProSa-web service obtained a
Z-score for each model [58,59]. In addition, Ramachandran plots were obtained with
the Discovery Studio v3.5 software [60] to visualize energetically allowed regions for
backbone dihedral angles ψ (psi) against ϕ (phi) of amino acid residues, and the favored
and allowed amino acids were determined and reported as a percentage [61]. Once all
monomers were validated in biomolecular in silico experiments, seven models of αβ-
tubulin of C. sativus were obtained with the guanosine triphosphate (GTP) and COL
ligands. All β-tubulin single models were merged into a heterodimer with α-tubulin
using the matchmaker tool for comparison from the UCSF Chimera v1.6 software [62] and
successively energy-minimized in the OPLS_2005 force field using the accessible version of
Maestro v9.6 software [61].

3.2. In Silico Docking Studies between Colchicine and the C. sativus αβ-Tubulin Model
The COL model was optimized in an entire energy minimization force field MMFF94
(Avogadro software v1.1.1) [63] and docked into the CBS of the crystallized model 4O2B,
and all seven validated αβ-tubulin models of C. sativus with Autodock v4.2 [64]. The
polar hydrogen atoms were configured with Kollman (AMBER) charge assignment for the
docking analysis preparation. A grid of 13.9 angstroms per side (37 points) was situated in
the colchicine binding site in the tubulin heterodimer. One hundred runs of a Lamarckian
genetic algorithm were configured. The Root Mean Square Deviation (RMSD) tolerance
of 2.0 Å was used to create the conformational cluster. The poses in the cluster with the
lowest energy (Gibbs free energy (∆Gb), and inhibition constant (Ki), were reported) were
analyzed. The Discovery Studio v3.5 software [60] was used to visualize and analyze
interactions in the poses network with the lowest binding energy at each model.
Additionally, the RMSD of each pose in the lowest energy cluster was obtained by
aligning to the conformation adopted by the COL crystallized conformation described by
Prota et al., 2014 [43] and published with PDB ID: 4O2B (COL4O2B ).

3.3. Technical Considerations before Starting the Phytotoxic Assay

3.3.1. Before Sowing
Cucumis sativus seeds were purchased from Germinal® (germinal.com.mx). Before
the test, an inspection was carried out, and using seeds and teguments with no damage
was essential to ensure a high percentage of germination. Briefly, seeds were washed with
neutral detergent and distilled water to remove polluting particles, additives maintaining
embryo viability, hormones for seed dormancy, as well as fungal growth prevention.
For C. sativus seed germination, Petri dishes (SYM, 75 × 15 mm) and filter paper as
a substrate were used, and the seeds (n = 25, in duplicate) were hydrated with purified
water (0.1 mL/cm2 ) and incubated in a manufactured incubator at room temperature
Plants 2022, 11, 1805 12 of 16

(26.3–31.4 ◦ C) in darkness [65]. Cucumber radicles were counted and photo-documented

every 12 h with a FinePix XP120 camera (FUJIFILM® , Villahermosa, Mexico).

3.3.2. After Sowing

The first day of germination (FDG) and the last day of germination (LDG) were
obtained; additionally, some germination factors were calculated [66–68]:
The germination percentage (%G) provides the viability of the seed lot (Equation (1)).
ni
%G = 100 (1)
N
where ni = number of seeds germinated at the time i and N = number of seeds sown.
The average germination time (T) measures the seeds’ average germination time to
germinate (Equation (2)).
∑ ( n i ti )
T= (2)
∑ ni
where ni = number of seeds germinated at the time i, and ti = number of days after sowing.
The half-time spread of germination (TSG) is the average time between the seed lot’s
first and last germination event (Equation (3)).

LDG − FDG
TSG = (3)
2
where LDG is the time (days) for the first visible radicle germinated and FDG is the time
(days) for the last observed radicle.
Treatment time (TT) is the germination time required to begin the phytotoxicity test
(Equation (4)).
TT = FDG + TSG (4)
In this work, the TT parameter was used to assess the acute phytotoxic effect of COL
on germinated seeds of C. sativus.

3.4. In Vitro Phytotoxic Effect of Colchicine

A stock was prepared with 1.05 mg/mL (2.5 mM) of COL (Sigma® Cat. C9754, ≥95%
purity HPLC powder). The volume to make serial dilutions (1:10) and the volume used in
Petri dishes (0.1 mL/cm2 ) for the treatment must be considered. Five exponential dilutions
(2.5 mM, 0.25 mM, 0.025 mM, 2.5 µM, and 0.25 µM) were tested to induce a concentration-
dependent phytotoxic effect by COL. In addition, a growth control (purified water) was
included in the assay.
Twenty-five cucumber seeds were sown in duplicate for each Petri dish (SYM, 75 × 15 mm).
COL treatment was initiated following the TSG. The radicles were photo-documented
before treatment initiation (zero hours) and subsequently at 24 and 48 h post-treatment.
The images were analyzed with GIMP 2 v10.6 software [69], and the root area in pixels was
measured for each radicle to obtain the average percentage of primary root growth (%RG)
(Equation (5)).
∑ Ax
%RG = × 100 (5)
∑ A0
where Ax is primary root area of treated seeds, A0 is primary root area of control seeds;
measured radicles (N) are in the same count in both treated and control seeds in the formula.
The data obtained at each concentration evaluated were normalized, and the first
ten data points, including defective radicles and non-germinated seeds, were removed.
Next, the average and standard deviation of the twenty-intermediate data (2nd and 3rd
quartiles) were calculated for the remaining forty measured radicles. Finally, the COL
logarithmic concentrations and %RG were graphed. A one-way analysis of variance
(ANOVA) with a Dunnett correction was performed, and a significant difference was
considered when p < 0.05 in a 95% confidence interval. In addition, the mean inhibitory
Plants 2022, 11, 1805 13 of 16

concentration (IC50 ) was assessed by interpolation (Y = 50%) in the straight-line equation

at the semilogarithmic graph.

4. Conclusions
In conclusion, the results presented in the current study indicated that the phytotoxic
effects of COL on cucumber root growth are dependent on the concentration, which
could be explained by the high conservation degree of the mitotic spindle proteins between
human and cucumber species (similarity = 100%). Furthermore, the CBS is situated between
the interface of α- and β-tubulin monomers, which, when docked with COL, has enough
affinity to form the colchicine-tubulin complex, promoting microtubule destabilization
and, as a macroscopic effect, a decrease in the primary root length from cucumber seeds,
a blebbing formation in the division zone of the radicles, as well as thickening of the
hypocotyl and inhibition of secondary roots. Additionally, the average concentration that
affects the morphology of the plant (0.85 mM and 1.77 mM at 24 and 48 h of exposure) was
described, together with the minimum tolerable concentration to prevent the growth of the
primary radicle (0.025 mM). Using COL as a herbicide could represent a soil risk due to its
antimitotic action mechanism, affecting plants and terrestrial and aquatic organisms.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/plants11141805/s1; Table S1: Identity percentages from the com-
parison of the α-tubulin sequences of C. sativus found in the UniProt database. Table S2: Identity
percentages from the comparison of the β-tubulin sequences of C. sativus found in the UniProt
database. Table S3: Alignment of α- and β-tubulin sequences of C. sativus against H. sapiens sequences
(P68363 for α-tubulin chain and Q9BVA1 for β-tubulin chain). Table S4. Interaction diagrams between
C. sativus tubulin models and the lowest-energy conformation of each cluster.
Author Contributions: Conceptualization, O.A.P.-M.; Formal analysis, L.C.-R.; Investigation, J.J.-P.;
Methodology, O.A.P.-M. and J.J.-P.; Resources, O.A.P.-M. and L.C.-R.; Validation, J.J.-P.; Visualization,
L.C.-R.; Writing—original draft, all authors; Writing—Review and Editing, O.A.P.-M. and M.R.-A. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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