Plant Cell Cultures as a Tool to Study Programmed Cell Death - Semantic Scholar
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
International Journal of
Molecular Sciences
Review
Plant Cell Cultures as a Tool to Study Programmed Cell Death
Massimo Malerba 1 and Raffaella Cerana 2, *
1 Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, 20126 Milan, Italy;
massimo.malerba@unimib.it
2 Dipartimento di Scienze dell’Ambiente e della Terra, Università degli Studi di Milano-Bicocca,
20126 Milan, Italy
* Correspondence: raffaella.cerana@unimib.it; Tel.: +39-02-6448-2932
Abstract: Programmed cell death (PCD) is a genetically controlled suicide process present in all living
beings with the scope of eliminating cells unnecessary or detrimental for the proper development of
the organism. In plants, PCD plays a pivotal role in many developmental processes such as sex deter-
mination, senescence, and aerenchyma formation and is involved in the defense responses against
abiotic and biotic stresses. Thus, its study is a main goal for plant scientists. However, since PCD
often occurs in a small group of inaccessible cells buried in a bulk of surrounding uninvolved cells,
its study in whole plant or complex tissues is very difficult. Due to their uniformity, accessibility,
and reproducibility of application of stress conditions, cultured cells appear a useful tool to investi-
gate the different aspects of plant PCD. In this review, we summarize how plant cell cultures can be
utilized to clarify the plant PCD process.
Keywords: cell cultures; programmed cell death; reactive oxygen species; reactive nitrogen species
Citation: Malerba, M.; Cerana, R.
1. Introduction
Plant Cell Cultures as a Tool to Study
Programmed Cell Death. Int. J. Mol. Programmed cell death (PCD) is a genetically controlled suicide process present in
Sci. 2021, 22, 2166. https://doi.org/ all living beings with the scope of eliminating cells unnecessary or detrimental for the
10.3390/ijms22042166 proper development of the organism. PCD plays a pivotal role in the plant lifestyle and it
is involved in several developmental (senescence, formation of tracheary elements, sex de-
Academic Editors: Robert Hasterok termination, aerenchyma formation, endosperm and aleuron maturation) and pathological
and Alexander Betekhtin contexts (response to stresses and to pathogen attack) [1,2]. Thus, its study is a main goal
for plant scientists. PCD process is organized in three phases. The first one is the induction
Received: 21 January 2021 phase, where the cells receive a wide range of extra- or intracellular signals (developmental
Accepted: 18 February 2021 input, pathogen attack, signals from neighboring cells, abiotic or biotic stresses). The second
Published: 22 February 2021 one is the effector phase, where the signals are elaborated to activate the death machinery.
The third one is the degradation phase, where the activity of the death machinery causes
Publisher’s Note: MDPI stays neutral the controlled destructuring of fundamental cell components [3]. The degradation phase
with regard to jurisdictional claims in shows a set of hallmarks that can be used to identify cells undergoing PCD. These hall-
published maps and institutional affil- marks include shrinkage of cellular and nuclear membrane, activation of specific cysteine
iations.
proteases called caspases, and activation of specific endonucleases able to cleave DNA in
controlled fragments (laddering) [3]. Unlike animals, where well-described forms of PCD
(for example apoptosis) are reported, in plants, the PCD process is still poorly understood
and the term PCD is widely used to describe cell death observed in different tissues and
Copyright: © 2021 by the authors. organs. At present, in plants, at least three forms of PCD have been described and cataloged
Licensee MDPI, Basel, Switzerland. on the basis of both cellular morphology and the main cellular compartment involved in
This article is an open access article the process. The “nucleus first form” is observable during the hypersensitive response to
distributed under the terms and pathogen attack and it is similar to animal apoptosis for the presence of specific hallmarks,
conditions of the Creative Commons
involvement of mitochondria included. The “chloroplast first form” is observable during
Attribution (CC BY) license (https://
foliar senescence, while the “vacuole first form” is observable during the maturation of
creativecommons.org/licenses/by/
vascular elements and during aerenchyma formation [4].
4.0/).
Int. J. Mol. Sci. 2021, 22, 2166. https://doi.org/10.3390/ijms22042166 https://www.mdpi.com/journal/ijmsInt.
Int. J.
J. Mol.
Mol. Sci.
Sci. 2021,
2021, 22,
22, 2166
2166 22 of
of 13
13
Int. J. Mol. Sci. 2021, 22, 2166 2 of 12
first
first form”
form” is
is observable
observable during
during foliar
foliar senescence,
senescence, while
while the
the “vacuole
“vacuole first
first form”
form” is
is observ-
observ-
able
able2.during
Mainthe
during the maturation of
maturationof
Advantages vascular
vascular elements
of Studying PCD inand
elements and during
Cellaerenchyma
during
Plant Cultures formation
aerenchyma formation [4].
[4].
2. The in-depth Studying study of the mechanism of plant PCD can often be a hard work as this
2. Main
Main Advantages
Advantages of of Studying PCD PCD in in Plant
Plant Cell
Cell Cultures
Cultures
process generally occurs in a small group of cells surrounded by the large number of
The
The in-depth
in-depth studystudy of of the
the mechanism
mechanism of of plant
plant PCD PCD can can often
often be be aa hard
hard work
work as as this
this
uninvolved cells
process present in whole plant and in complex tissues. number
For a number of reasons,
process generally
generally occurs
occurs in in aa small
small group
group of of cells
cells surrounded
surrounded by by the
the large
large number of of un-
un-
cultured
involved cells appeared an attractive model to several plant biologists to face this matter.
involved cellscells present
present in in whole
whole plant plant and
and in in complex
complex tissues.tissues. ForFor aa number
number of of reasons,
reasons,
For
cultured example,
cells large
appeared quantities
an attractive of cell
model material
to several can be
plant easily
biologists
cultured cells appeared an attractive model to several plant biologists to face this matter. maintained
to face or
this quickly
matter. generated
Forfrom
For example,
example,stocks toquantities
large
large furnish investigators
quantities of
of cell materialwith
cell material can
can be a great
be easilynumber
easily maintained
maintained of fast-dividing
or
or quickly
quickly gener-
gener- and relatively
ated from stocks to furnish investigators with a great number of fast-dividing and rela-good system
ated homogeneous
from stocks to cells.
furnish In addition,
investigators the greater
with a accessibility
great number of makes cell
fast-dividing cultures
and a
rela-
tively
to homogeneous
tively which different
homogeneous cells.
cells. In
In addition,
compounds
addition,or the greater
thestress
greater accessibility
conditions
accessibility can makes cell
cell cultures
be easily
makes furnished
cultures aa good
goodto or removed
system
system to which different compounds or stress conditions can be easily furnished to or
from. to which
Finally,different
with compounds
vital dyes suchor stress
as Evansconditions
Blue can
or be easily
fluorescein furnished
diacetate, to it
or is relatively
removed
simple to follow the growth or death response of cells to different stressors is
removed from.
from. Finally,
Finally, with
with vital
vital dyes
dyes such
such as
as Evans
Evans Blue
Blue or
or fluorescein
fluorescein diacetate,
diacetate, it
it is
by removing
relatively
relatively simple
simple to
to follow
follow the
the growth
growth or
or death
death response
response of
of cells
cells
them at time intervals from the culture batches or by observing the fate of single cells in to
to different
different stressors
stressors by
by
removing
removing them
them Toat
at time
time intervals
intervals from
from the
the culture
culture batches
batches or
or by
by observing
observing the
the fate
fate of
of single
single
real time. further characterize the PCD process, it is relatively easy to assess under
cells in
in real
cellsthe real time.
time. To
To further
further characterize
characterize the
the PCD
PCD process,
process, it
it is
is relatively
relatively easy
easy to
to assess
assess
microscope any visible morphological changes that occur in cultured cells induced
under
under thethe microscope
microscope any any visible
visible morphological
morphological changes that
that occur
occur in cultured
cultured cells
cells in-
duced
to undergo
to undergo
PCD PCD
[5].
[5].
For
For
example,
example,
nucleuschanges
nucleus
condensation,
condensation,
a in
modification in-
often occurring
duced to undergo PCD [5]. For example, nucleus 0 0 condensation, a modification often oc-
a modification often oc-
during
curring PCD, is easily observable with 4 ,6 -diamidino-2-phenylindole (DAPI). Thus, a large
curring during
during PCD,PCD, is is easily
easily observable
observable with with 4’,6’-diamidino-2-phenylindole
4’,6’-diamidino-2-phenylindole (DAPI). (DAPI).
Thus,set of
Thus, aa large
cell
large set
cultures,
set of
of cell
in
cell cultures,
particular
cultures, in in particular
(but
particular (but
not
(but not
only)
not only)
those
only) those
obtained
those obtained
obtained from
from
from the
the
the model
model
model plants
Arabidopsis
plants
plants Arabidopsis
Arabidopsis thaliana
thalianaand
thaliana and Nicotiana
and Nicotiana tabacum,
Nicotiana tabacum,
tabacum, hashas
has been
beenbeen proposed
proposed
proposed and and utilized
and utilized
utilized to
to inves-
inves-to investigate
plant
tigate
tigate plant
plantPCD PCD[5].
PCD [5].After
[5]. After some
After some earlyindications
some early
early indications
indications ([6]
([6]
([6] and
andand references
references
references therein),
therein),
therein), in theinlast
in the the last years,
last
several
years,
years, several
several different
differentconditions
different conditions have
conditions have beenproven
have been
been provenable
proven able
able to to
to induce
induce
induce PCD
PCD PCDin in cell
in cell
cell cultures. In this
cultures.
cultures.
In
In this
this review,
review,
review, wewe
we summarize
summarize
summarizethe the literature
literature on
theliterature onPCD
on PCDinduced
PCD induced
induced by
byby biotic and
biotic
biotic andandabiotic stresses
abiotic
abiotic stressesstresses useful
useful
useful to
to clarify
to clarify
clarifythethe
the process
process
processin in plants.
inplants.
plants.
3.
3. PCD Induced
3. PCD
PCD in
in Cell
Induced
Induced inCultures
Cell by
by Biotic
Cell Cultures
Cultures byStress
Biotic Biotic Stress
Stress
Several
Several toxins
toxins and
and metabolic
metabolic products
products
Several toxins and metabolic products obtained by
by microorganisms
obtainedobtained
microorganisms and
and fungi
fungi can
by microorganisms can
and fungi can
induce
induce PCD
PCD inin cell
cell cultures,
cultures, as
as summarized
summarized in
in Table
Table 1.
1.
induce PCD in cell cultures, as summarized in Table 1.
Table
Table 1.
1. Biotic
Table Biotic programmed
programmed
1. Biotic cell
cell death
programmed cell (PCD)
death deathinducers
(PCD) inducers in
in plant
plant cell
(PCD) inducers cell cultures.
cultures.
in plant cell cultures.
Main
Main Characteristics
Characteristics of
of Induced
Induced
Plant
Plant Species
Species PCD Induced
Induced by
PCDInduced by Reference
Reference
Plant Species PCD by Main Characteristics
PCD
PCD of Induced PCD Reference
H
H O22 2accumulation,
H222O
O accumulation,
accumulation, changes
changes
changes in cell in cell
in cell
Acer
Acer pseudoplatanus
Acer pseudoplatanus
pseudoplatanus L.
L. L. and
and and nucleus morphology,
nucleus
nucleus DNA
morphology,
morphology, DNA [7]
[7] [7]
fragmentation
DNA fragmentation
fragmentation
Tunicamycin
Tunicamycin
Tunicamycin
H
H O22 2accumulation,
H222O
O accumulation,
accumulation, changes
changes
changes in cell in cell
in cell
Acer
Acer pseudoplatanus
Acer pseudoplatanus
pseudoplatanus L. L.
L. and and
and nucleus
nucleus
nucleus morphology,
morphology,
morphology, DNA
DNA [7]
[7] [7]
fragmentation
DNA fragmentation
fragmentation
Int. J. Mol. Sci. 2021, 22, 2166 3 of 13
Brefeldin
Brefeldin AA
Brefeldin A
H2O2 accumulation, changes in cell
Acer pseudoplatanus
Acer pseudoplatanus L. L. andH 2 O2 morphology,
nucleus accumulation,
DNA changes
[8] in cell and
[8]
nucleus morphology, DNA laddering
laddering
Fusicoccin
Fusicoccin
Activation of gene transcription and
Arabidopsis thaliana (L.) Heynh. protein synthesis, DNA [9]Int. J. Mol. Sci. 2021, 22, 2166 3 of 13
H2O2 accumulation, changes in cell
Acer pseudoplatanus L. and nucleus morphology, DNA [8]
laddering
H2O2 accumulation, changes in cell
Acer pseudoplatanus L. and nucleus morphology, DNA [8]
Int. J. Mol. Sci. 2021, 22, 2166 laddering
3 of 12
H2O2 accumulation, changes in cell
Acer pseudoplatanus L. and nucleus morphology, DNA [8]
laddering
Fusicoccin
Table 1. Cont.
Plant Species PCD Fusicoccin
Induced by Main Characteristics of Induced PCD Reference
Fusicoccin
Activation of gene transcription and
Arabidopsis thaliana (L.) Heynh. protein synthesis, DNA [9]
Activation of gene transcription and
Arabidopsis thaliana fragmentation
Arabidopsis thaliana (L.)
(L.) Heynh. Activation of gene transcription
protein synthesis, DNA [9] and
[9]
Heynh. protein synthesis, DNA fragmentation
fragmentation
Activation of gene transcription and
Arabidopsis thaliana (L.) Heynh. protein synthesis, DNA [9]
fragmentation
Thaxtomin AA
Thaxtomin
Nicotiana tabacum L. cv. Bright Thaxtomin
Metabolic products present A
in the Alternaria alternata culture Cytoplasm shrinkage, chromatin
Nicotiana tabacum [10]
Nicotiana
Yellow 2 L.
tabacum cv.Bright Metabolic
L. cv. Metabolic products
products present presentalternata
in the Alternaria
Thaxtomin
filtrate A in theculture Cytoplasm
Cytoplasm shrinkage,
shrinkage, DNA
condensation, chromatin chromatin
laddering[10]
Yellow
Nicotiana 2
tabacum L. cv. Bright Metabolic products presentfiltrate
in the Alternaria alternata culture condensation,
Cytoplasm DNA laddering
shrinkage, chromatin [10]
Bright Yellow 2Yellow 2
Alternaria alternata culture filtrate
filtrate
condensation, DNA laddering
condensation, DNA laddering
[10]
HH2OO2 accumulation,
2 H accumulation,
2 2O2 accumulation,
lipid lipid peroxidation,
lipid
Nicotiana tabacum L.L. cv. H2O2 accumulation, lipid
NicotianaNicotiana
tabacum tabacum
L. cv.
Nicotiana NC89cv. L.
tabacum NC89
cv. NC89 caspase-3-like protease activity,
peroxidation, caspase-3-like
peroxidation,
peroxidation, caspase-3-like proteaseprotease
caspase-3-like [11]
[11] protease
[11] [11]
NC89 activity,
activity, mitochondrial
mitochondrial
activity, dysfunction
dysfunction
mitochondrial dysfunction
mitochondrial dysfunction
Fusaric acid
Fusaric acid
Fusaric acid
Changes in vacuole shape,
Nicotiana tabacum L. cv.L. cv. Bright
Nicotiana tabacum Fusaric acid Changes in vacuole shape, endoplasmic
Changes infilaments
vacuole shape, [12]
Nicotiana tabacumYellow
L. cv. 2Bright Culture filtrates
Culture filtratesof Erwinia
of Erwinia carotovora
carotovora endoplasmic actin [12]
Bright Yellow
Yellow 2 2
Nicotiana tabacum L. cv. Bright
Culture filtrates of Erwinia carotovora actin filaments disassembly
Changes
endoplasmic
disassembly in filaments
actin vacuole shape, [12]
Culture filtrates of Erwinia carotovora disassemblyactin filaments
endoplasmic [12]
Yellow 2
disassembly
Nicotiana tabacum L. cv. Bright H O accumulation, activation of
H2O2 accumulation, activation of
2 2
Nicotiana tabacum L. cv. gene transcription, mitochondrial [13]
Yellow 2 H2O2dysfunction gene transcription,
accumulation, activation of [13]
Bright Yellow
Nicotiana tabacum2L. cv. Bright
Int. J. Mol. Sci. 2021, 22, 2166 gene transcription, mitochondrial dysfunction
[13] 4 of 13
Yellow
Int. J. Mol. Sci. 2021, 2
22, 2166 mitochondrial 4 of 13
H2O2 dysfunction
accumulation, activation of
Nicotiana tabacum L. cv. Bright
gene transcription, mitochondrial [13]
Yellow 2
dysfunction
Deoxynivalenol
Deoxynivalenol
Deoxynivalenol
Int. J. Mol. Sci. 2021, 22, 2166 4 of 13
Deoxynivalenol
Generationof of
CaCa
2+ transient, H2O2
H22+
Arabidopsis
Arabidopsis thaliana
thaliana
Arabidopsis (L.)
thaliana (L.)
Heynh.
(L.) Heynh. Generation
Generation of Ca
2+ transient, O2 transient,
[14] [14]
accumulation
accumulation [14]
Heynh. H2 O2 accumulation
Generation of Ca2+ transient, H2O2
Arabidopsis thaliana (L.) Heynh. [14]
accumulation
Ceramides
Ceramides
Ceramides
ROS
ROS and
ROSandethylene
and accumulation,
ethylene
ethylene accumulation,
accumulation,
protoplast
protoplast shrinkage,
shrinkage, nucleus
nucleus nucleus
Solanum
Solanum lycopersicum
lycopersicum L.L. Phosphatidic
Ceramides acid
Phosphatidic acid protoplast shrinkage, [15] [15]
Solanum lycopersicum L. Phosphatidic acid ROS and condensation,
condensation,
ethylene caspase-3-like
accumulation,caspase-3-likeprotease
protease [15]
protoplast shrinkage,condensation,
activity
nucleus activity
caspase-3-like
Solanum lycopersicum L. Phosphatidic acid [15]
condensation, caspase-3-like proteaseprotease activity
activity
Nicotiana tabacum L. cv. Bright ROS accumulation, caspase-3-like
Nicotiana tabacum
Nicotiana tabacumL. cv. Bright ROScaspase-3-like
accumulation, caspase-3-like
Yellow 2 L. Yellow
cv. L. cv. Bright ROS accumulation, caspase-3-like
Nicotiana tabacum ROS accumulation, [16]
2 protease activity
[16]
protease activity [16] [16]
BrightYellow
Yellow 2
2 protease activity
protease activity
Acrolein
Acrolein
Acrolein
Acrolein
Cell shrinkage, chromatin
Cell shrinkage, chromatin
Nicotiana tabacum L.tabacum
Nicotiana cv. L. cv. Bright condensation, and nuclear DNA [17]
Yellow 2 condensation, and nuclear [17]
Bright Yellow 2 degradation
DNA degradation
Cell shrinkage, chromatin
Nicotiana tabacum L. cv. Bright
condensation, and nuclear
Cell shrinkage, DNA
chromatin [17]
Yellow
Nicotiana tabacum L. 2cv. Bright
degradation
condensation, and nuclear DNA [17]
Yellow 2
degradation
Narciclasin
Narciclasin
Narciclasin
ROS and RNS accumulation, changes
Populus alba L. Narciclasin in cell and nucleus morphology, [18]Cell shrinkage, chromatin
Nicotiana tabacum L. cv. Bright
condensation, and nuclear DNA [17]
Yellow 2
Int. J. Mol. Sci. 2021, 22, 2166 degradation 4 of 12
Table 1. Cont.
Plant Species PCDNarciclasin
Induced by Main Characteristics of Induced PCD Reference
ROS
ROS andand RNS accumulation,
RNS accumulation, changes changes in
Populus
Populus albaalbaL.L. in cell cell
and nucleus morphology,
and nucleus [18]
morphology, [18]
chromatin condensation
chromatin condensation
Int. J. Mol. Sci. 2021, 22, 2166 5 of 13
Int. J. Mol. Sci. 2021, 22, 2166 5 of 13
Int.Int. J. Mol.
J. Mol. Sci.Sci. 2021,
2021, 22,22, 2166
2166 5 of 13 13
5 of
Medicagenic acid
Medicagenic acid
Nicotiana tabacum L. cv. Bright ROS accumulation, DNA
Nicotiana tabacum
Nicotiana
Nicotiana tabacum
Yellow 2L.
tabacum
L. cv.
cv.L. cv. Bright
Bright ROSROS ROSandaccumulation,
accumulation, DNA DNA
accumulation,
fragmentation [19] DNA
hypomethylation
[19]
Nicotiana tabacum
Yellow Yellow
2
L. 2cv. Bright ROS accumulation,
fragmentation DNA
and hypomethylation
fragmentation and hypomethylation[19]
[19] [19]
Bright Yellow Yellow 22 fragmentation and
fragmentation and hypomethylationhypomethylation
Juglone
Juglone
Juglone
Juglone
Juglone DNA fragmentation, expression of
DNA fragmentation, expression of
Vitis labrusca L. Alanine DNA
DNA
DNA
defense-related fragmentation,
genes,
fragmentation, accumulation
fragmentation,expression expression of
of [20]
expression of
Vitis labrusca L. Alanine defense-related genes, accumulation [20]
Vitis labrusca
VitisVitis L.L. L.
labrusca
labrusca Alanine
Alanine
Alanine defense-related
of phenolic genes,
defense-related
defense-relatedcompounds genes, accumulation
accumulation
genes, accumulation
of phenolic compounds
[20] [20] of [20]
of phenolic compounds
phenolic compounds
of phenolic compounds
Changes in nucleus morphology,
Arabidopsis thaliana (L.) Heynh. DNAChanges
laddering,inactivation
nucleus of gene
morphology, [21]
Changes in nucleus morphology,
Arabidopsis thaliana
Arabidopsis (L.)(L.)
thaliana Heynh.
Heynh.
Changes
Changes
DNA
DNA laddering,
laddering,
in activation
in nucleus
transcription nucleus
activation ofmorphology,
morphology,
of genegene [21] [21] DNA
Arabidopsis
Arabidopsis thaliana
thaliana (L.)(L.)
Heynh. DNA laddering, activation activation
of gene [21]
laddering,
transcription
transcription of [21]
Heynh. transcription
gene transcription
6-benzylaminopurine
6-benzylaminopurine
6-benzylaminopurine
6-benzylaminopurine
6-benzylaminopurine
Cell shrinkage, nuclear DNA
degradation, mitochondrial
Arabidopsis thaliana (L.) Heynh. [22]
Cell shrinkage,
dysfunction, inductionnuclear DNA
of caspase-
Cell shrinkage,
Cell shrinkage, nuclear DNA nuclear DNA
degradation,
Cell like mitochondrial
activity
shrinkage,mitochondrial
nuclear DNA [22]
Arabidopsis thaliana (L.)
Arabidopsis (L.) Heynh.
Arabidopsisthaliana dysfunction,degradation, mitochondrial
degradation,
thaliana (L.) Heynh. induction of caspase- [22]
degradation,
dysfunction, mitochondrial
induction of caspase- [22]
Heynh.
Arabidopsis thaliana (L.) Heynh. dysfunction,
like
dysfunction,like
activity
induction induction[22]of
activity of caspase-
likecaspase-like
activity activity
Acetylsalicilic acid
Acetylsalicilic acid
Acetylsalicilic acid
Acetylsalicilic acid
Acetylsalicilic acid
ROS and RNS accumulation, nuclear
DNA degradation, caspase-3-like
Acer pseudoplatanus L.
ROS
protease
ROS and RNSand RNS accumulation,
activity, mitochondrial
accumulation, nuclear
[23] nuclear
dysfunction, expression of defense-
Acer pseudoplatanus L.
DNA
DNA degradation,
ROS and
degradation,
RNSgenes
related
caspase-3-like caspase-3-like
accumulation, nuclear
protease activity, mitochondrialnuclear[23]
Acer pseudoplatanus L. ROS
DNAandprotease activity,
RNS accumulation,
degradation, mitochondrial
caspase-3-like [23]
Acer pseudoplatanus L. dysfunction,
DNA expression caspase-3-like
degradation, of defense-
Acer pseudoplatanus L. protease dysfunction, expression
activity, mitochondrial
related genes
[23]of
protease activity,
dysfunction, mitochondrial
expression of defense- [23]
dysfunction, defense-related
expression genes
related genesof defense-
related genes
Chitosan
For example, in Acer pseupdoplatanus L.-cultured cells, tunicamycin, an inhibitor of N-
Chitosan
linked protein glycosylation
Chitosan produced by Streptomyces lysosuperificus, and brefeldin A, an
inhibitor of protein trafficking from the Golgi apparatus produced by Eupenicillium bre-
For example, in Acer pseupdoplatanus L.-cultured cells, tunicamycin, an inhibitor of N-
feldianum, induce a Chitosan
PCD with apoptotic features such as reactive oxygen species (ROS)
linkedForprotein glycosylation
example, Chitosan produced
in Acer by Streptomyces lysosuperificus,
pseupdoplatanus L.-cultured and brefeldin
cells, A, an
tunicamycin, an inhibitor of
For example,
inhibitor intrafficking
of protein Acer pseupdoplatanus L.-cultured
from the Golgi apparatuscells, tunicamycin,
produced an inhibitor
by Eupenicillium bre-of N-
N-linked
linked Forprotein
feldianum,
protein
example, in glycosylation
Acer pseupdoplatanus
glycosylation
induce a PCD withproduced
apoptoticby
produced
L.-cultured
Streptomyces
features
by Streptomyces
cells, tunicamycin,
such aslysosuperificus,
reactive oxygen
an
and
lysosuperificus,
inhibitor
brefeldin
species
of
(ROS)A, an
N- and brefeldin A,
linked protein
inhibitor glycosylation
of protein produced
trafficking byGolgi
from the Streptomyces lysosuperificus,
apparatus produced by and brefeldin A,bre-
Eupenicillium an
inhibitor
feldianum,ofinduce
protein
a trafficking from the Golgi
PCD with apoptotic apparatus
features such as produced by Eupenicillium
reactive oxygen bre-
species (ROS)
feldianum, induce a PCD with apoptotic features such as reactive oxygen species (ROS)Int. J. Mol. Sci. 2021, 22, 2166 5 of 12
an inhibitor of protein trafficking from the Golgi apparatus produced by Eupenicillium
brefeldianum, induce a PCD with apoptotic features such as reactive oxygen species (ROS)
accumulation, changes in cell and nucleus morphology, and specific DNA fragmenta-
tion [7]. In the same experimental material, fusicoccin a well-known activator of the plasma
membrane H+ -ATPase produced by Phomopsis amygdali induces PCD with similar character-
istics [8]. The well-identified target of these molecules permitted to test the role of specific
cell compartments or physiological functions in the induction, development, and execution
of plant PCD process. In particular, investigation conducted with fusicoccin showed that
the phytotoxin-induced PCD involves changes in actin cytoskeleton [24] and utilizes the
plant hormone ethylene as regulative molecule in addition to ROS and reactive nitrogen
species (RNS) [25]. Interestingly, inhibition of cytochrome c release from the mitochondrion
by cyclosporin A markedly prevents the fusicoccin-induced PCD [26], and recently a possi-
ble role as signaling molecule for peroxynitrite has been proposed [27]. These results also
sustain the fundamental role of cytochrome c and peroxynitrite in the induction of PCD
process in plants. In Arabidopsis thaliana cultures, thaxtomin A, an inhibitor of cellulose
biosynthesis produced by Streptomyces scabiei, induces a PCD dependent on active gene
transcription and de novo protein synthesis and that displays apoptotic-like features such
as specific DNA fragmentation [9]. Interestingly, addition of auxin to Arabidopsis cell cul-
tures prevents thaxtomin-induced PCD possibly by stabilizing the plasma membrane–cell
wall–cytoskeleton continuum [28]. In tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow
2) cell suspensions metabolic products present in the Alternaria alternata culture filtrate
induce a PCD dependent on ROS generation that shows cytoplasm shrinkage, chromatin
condensation, and DNA laddering [10]. Interestingly, the PCD induced in tobacco BY-2
cells by Pectobacterium carotovorum and Pectobacterium atrosepticum is reduced by culture
filtrate of non-pathogenic Streptomyces sp. OE7 that through cytosolic Ca2+ changes and
generation of ROS induces defense responses [29]. This highlights the complexity of the
interactions between microorganisms and plants and the need for further investigations.
In tobacco cv. NC89-cultured cells, fusaric acid, a non-specific toxin produced mainly by
Fusarium spp., causes PCD with mechanism that is not well understood that, however,
involves ROS overproduction and mitochondrial dysfunction [11]. In fact, pre-treatment
of tobacco cells with the antioxidant molecule ascorbic acid and with the nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenyl iodonium signifi-
cantly reduces the fusaric acid-induced accumulation of dead cells as well as the increase
in caspase-3-like protease activity. Moreover, oligomycin and cyclosporine A, inhibitors
of the mitochondrial ATP synthase and the mitochondrial permeability transition pore,
respectively, also reduce the rate of fusaric acid-induced cell death [11]. PCD induced in cell
cycle-synchronized tobacco BY-2 cells by application of culture filtrates of Erwinia carotovora
involves changes in vacuole shape and disassembly of endoplasmic actin filaments [12].
In tobacco BY-2 cultures, deoxynivalenol, a mycotoxin synthesized by Fusarium culmorum
and Fusarium graminearum, induces a PCD sustained by different cross-linked pathways
involving ROS generation linked, at least partly, to a mitochondrial dysfunction and to tran-
scriptional downregulation of the alternative oxidase (Aox1) gene and showing regulation
of ion channel activities participating in cell shrinkage [13]. Interestingly, this mycotoxin
is also able to induce PCD in animal cells, but with different characteristics. This sug-
gests the presence of different ways to induce PCD between animals and plants (original
articles cited in [13]). Some metabolites able to induce PCD in plant cultured cells can
originate from the degradation of cellular components or are produced by the primary and
secondary metabolism of microorganisms and plants. For example, ceramides, lipids de-
rived from the membranes of eukaryotic cells, can induce PCD in Arabidopsis cultures in a
Ca2+ -dependent manner. In fact, the calcium channel-blocker lanthanum chloride substan-
tially reduces the amount of ceramide-induced cell death [14]. Interestingly, in the same
material, sphingolipids can reduce apoptotic-like PCD induced by different treatments,
ceramides and heat stress included [30]. Moreover, in tomato suspensions, cell death
induced by camptothecin, fumonisin B1, and CdSO4 is regulated by phosphatidic acid.Int. J. Mol. Sci. 2021, 22, 2166 6 of 12
This cell death involves ROS and ethylene, depends on caspase-like proteases, and ex-
presses morphological features of apoptotic-like PCD such as protoplast shrinkage and
nucleus condensation [15]. Reactive carbonyl species (namely, acrolein, shown in Table 1)
derived from lipid peroxidation can activate caspase-3-like proteases to initiate PCD in
tobacco BY-2 cultures [16]. In the same experimental material, narciclasine (NCS), a plant
growth inhibitor isolated from the secreted mucilage of Narcissus tazetta bulbs, can induce
typical PCD-associated morphological and biochemical changes, namely, cell shrinkage,
Int. J. Mol. Sci. 2021, 22, 2166 7 of 13
chromatin condensation, and nuclear DNA degradation [17]. Among primary and sec-
ondary metabolites, the triterpene saponins (namely, medicagenic acid, shown in Table 1)
protoplastsativa)
from alfalfaas(Medicago applied
shrinkage Populuscondensation
and tonucleus alba cell cultures induce acarbonyl
[15]. Reactive PCD dependent
species
on RNS and ROS acrolein,
(namely, production shown and showing
in Table changes
1) derived in nucleus
from lipid morphology
peroxidation can activate and chro-
caspase-
3-like proteases
matin condensation to initiate
[18]. In tobaccoPCD BY-2in cultures,
tobacco BY-2 cultures
juglone [16]. In the same experimental
(5-hydroxy-1,4-naphthoquinone)
causes cellmaterial,
death withnarciclasine (NCS), a plant growth
ROS overproduction inhibitor isolated
accompanied from the secreted
by formation mucilage
of apoptic-like
of Narcissus tazetta bulbs, can induce typical PCD-associated morphological and biochem-
nuclear bodies (indication of DNA fragmentation) and DNA hypomethylation [19]. In Vitis
ical changes, namely, cell shrinkage, chromatin condensation, and nuclear DNA degrada-
labrusca suspension cultures, L-alanine is the only amino acid able to induce PCD accom-
tion [17]. Among primary and secondary metabolites, the triterpene saponins (namely,
panied by medicagenic
DNA fragmentation,
acid, shown in expression of alfalfa
Table 1) from defense-related genes,
(Medicago sativa) andtoaccumulation
applied Populus alba
of phenoliccell
compounds [20].
cultures induce Plant
a PCD phytoregulators
dependent on RNS and ROScan also activate
production andPCD
showingin plant
changes cell
cultures. For
in example, high levelsand
nucleus morphology of cytokinins (namely, 6-benzylaminopurine,
chromatin condensation shown in
[18]. In tobacco BY-2 cultures,
juglone
Table 1) induce PCD(5-hydroxy-1,4-naphthoquinone) causes cell death
in Arabidopsis cultures by accelerating with ROSprocess
a senescence overproduction
character-
ized by DNA accompanied
ladderingby andformation
expression of ofapoptic-like nuclear bodies
specific senescence markers(indication
[21]. InoftheDNA
same
fragmentation) and DNA hypomethylation [19]. In Vitis labrusca suspension cultures, L-
material acetylsalicylic acid, a derivative from the plant hormone salicylic acid induces typ-
alanine is the only amino acid able to induce PCD accompanied by DNA fragmentation,
ical PCD-linked morphological
expression and genes,
of defense-related biochemical changes,of
and accumulation namely,
phenoliccell shrinkage,
compounds [20].nuclear
Plant
DNA degradation, mitochondrial membrane potential, cytochrome c
phytoregulators can also activate PCD in plant cell cultures. For example, high levelsfrom
loss of release of
mitochondria, and induction
cytokinins of caspase-like activity
(namely, 6-benzylaminopurine, shown[22]. Finally,
in Table in Acer
1) induce PCD pseudoplatanus
in Arabidopsis
cultures, chitosan,
cultures bytheaccelerating
non-toxic aand inexpensive
senescence processcompound
characterized obtained
by DNAby deacetylation
laddering and ex- of
chitin, the pression of specific senescence
main component markers [21].
of the exoskeleton ofIn the same material
arthropods as well acetylsalicylic
as of the cell acid, a
walls
derivative
of many fungi, from the
induces a PCDplantmediated
hormone salicylic
by ROS acidand
induces
RNStypical PCD-linkedand
accumulation morpholog-
showing
ical and biochemical changes, namely, cell shrinkage, nuclear DNA degradation, loss of
changes in gene expression and specific DNA fragmentation [23].
mitochondrial membrane potential, cytochrome c release from mitochondria, and induc-
tion of caspase-like activity [22]. Finally, in Acer pseudoplatanus cultures, chitosan, the non-
4. PCD Induced in Cell Cultures by Abiotic Stress
toxic and inexpensive compound obtained by deacetylation of chitin, the main component
Severalof abiotic stresses
the exoskeleton of ranking
arthropodsfrom different
as well as of thechemicals
cell walls of such
many as heavy
fungi, metals
induces a PCDand
mediated
dyes to ambient by ROS
growth and RNS accumulation
conditions can induce PCD and showing
in plantchanges in gene expression
cell cultures, and
as summarized
in Table 2. specific DNA fragmentation [23].
Table4.2.PCD Induced
Abiotic PCDin Cell Cultures
inducers bycell
in plant Abiotic Stress
cultures.
Several abiotic stresses ranking from different chemicals such as heavy metals and
Plant Species dyes Induced
PCD to ambient
bygrowth conditions Main
can induce PCD in plant
Characteristics ofcell cultures,
Induced PCD as summarized
Reference
in Table 2.
Changes in cell and nucleus
Nicotiana tabacum L. cv.
Cadmium ions morphology, appearance of
Table 2. Abiotic PCD inducers in plant cell cultures.
[31]
Bright Yellow 2
autophagic bodies
Plant Species PCD Induced by Main Characteristics of Induced PCD Reference
Nicotiana
Caspase-like protease activity,
Nicotiana tabacum L. cv.tabacum L. Cadmium ions
Changes in cell and nucleus morphology, appearance of
[31]
cv. Bright Yellow 2 Aluminium oxide nanoparticles mitochondrial dysfunction,
autophagic bodies [32]
Bright Yellow 2
Nicotiana tabacum L. DNA fragmentation
Caspase-like protease activity, mitochondrial
Aluminium oxide nanoparticles [32]
cv. Bright Yellow 2 dysfunction,
Changes DNA fragmentation
in cell and nucleus
Changes in cell and nucleus
morphology, DNA morphology, DNA
fragmentation,
Viola tricolor L.
Viola tricolor L. Zinc
Zincand
and lead ions
lead ions fragmentation, caspase-like and papain-like cysteine [33]
[33]
caspase-like and papain-like cysteine
protease activity
protease activity
Nicotiana
ROS accumulation, lipid peroxidation,
Nicotiana tabacum L. cv.tabacum L. ROS accumulation, lipid peroxidation, caspase-3-like
[34]
cv. Bright Yellow 2 caspase-3-like
protease protease
activity, DNA activity,
fragmentation [34]
Bright Yellow 2
DNA fragmentation
Fluoranthene
FluorantheneInt. J. Mol. Sci. 2021, 22, 2166 7 of 12
Int. J. Mol. Sci. 2021, 22, 2166 8 of 13
Int. J. Mol. Sci. 2021, 22, 2166 8 of 13
Table 2. Cont.
Plant Species PCD Induced by Main Characteristics of Induced PCD Reference
Arabidopsis thaliana ROS accumulation, lipid peroxidation, specific gene
Arabidopsis thaliana ROS accumulation, lipid peroxidation, specific gene [35]
Arabidopsis thaliana
(L.)(L.)
Heynh. ROS accumulation,
activation lipid peroxidation, [35]
[35]
Heynh. (L.) Heynh. specificactivation
gene activation
Rose
RoseBengal
Bengal
Rose Bengal
Glycine max (L.)
Mitochondrial dysfunction, DNA
Mitochondrial dysfunction, DNA fragmentation,
Glycine max (L.) Glycine
Merr. max (L.) fragmentation,
Mitochondrial dysfunction, caspase-3-like
DNA fragmentation, [36]
[36]
Merr. caspase-3-like protease activity [36]
Merr. protease
caspase-3-like activity
protease activity
Dinitro-o-cresol
Dinitro-o-cresol
Populus euphratica Dinitro-o-cresol Cell shrinkage,
Cell shrinkage, chromatin chromatin
condensation, nuclear DNA
Populus euphratica 1-Butanol Celldegradation,
shrinkage, chromatin condensation, nuclear DNA [37]
Oliv. 1-Butanol caspase-3-like
condensation, protease DNA
nuclear activity [37]
Populus euphratica Oliv.
Oliv.
Populus euphratica
1-Butanol degradation,
Elevation caspase-3-like
of cytosolic Ca protease
2+ levels, ROS activity
accumulation,
[37]
ATP (externally added) degradation, caspase-3-like [38]
Populus euphratica
Oliv. Elevation ofmitochondrial levels, ROS accumulation,
cytosolic Ca2+dysfunction
ATP (externally added) protease activity [38]
Oliv. mitochondrial
ROS accumulation, cytoplasmicdysfunction
shrinkage, DNA
Nicotiana tabacum L. Elevation
ROS accumulation,
fragmentation, of cytosolic
cytoplasmic
caspase-3-like Ca2+activity,
levels,
shrinkage,
protease DNA
Populus euphratica Oliv.Yellow
Nicotiana tabacum2 L. Heat stress added)
ATP (externally fragmentation, ROS accumulation,
caspase-3-like protease activity, [39]
[38]
cv. Bright Heat stress mitochondrial dysfunction, induction of defense-related [39]
cv. Bright Yellow 2 mitochondrial mitochondrial
dysfunction,
genes dysfunction
induction of defense-related
Cakile maritime Scop. ROS accumulation, lipid genes
peroxidation, specific gene
ROS accumulation, cytoplasmic
Cakile maritime
Arabidopsis Scop.
thaliana Salt stress ROS accumulation,
activation, caspase-3-likelipid peroxidation,
protease specific gene
activity, mitochondrial [40]
Arabidopsis
shrinkage, protease
DNA fragmentation,
Nicotiana tabacum cv. thaliana
L. Heynh.
(L.) Salt stress activation, caspase-3-like
dysfunction activity, mitochondrial [40]
Heat stress caspase-3-like protease activity, [39]
Bright Yellow 2(L.) Heynh.
Arabidopsis thaliana ROS accumulation, Ca dysfunction
2+ influx, changes in cell and
Arabidopsis thaliana Ozone mitochondrial
ROS accumulation, dysfunction, induction [41]
nucleusCamorphology
2+ influx, changes in cell and
(L.) Heynh. Ozone [41]
(L.) Heynh. ofnucleus
defense-related genes
morphology caspase-3-like
ROS accumulation, DNA fragmentation,
Vitis vinifera L. Darkness (in senescent cultures) ROSprotease
accumulation, DNA fragmentation, caspase-3-like [42]
Vitis vinifera L. Darkness (in senescent cultures) ROSactivity,
accumulation,
mitochondriallipiddysfunction
peroxidation, [42]
Cakile maritime Scop. protease activity, mitochondrial dysfunction
Nicotiana tabacum L. specific gene
ROS accumulation, DNAactivation,
fragmentation, caspase-3-like
mitochondrial
Arabidopsis thaliana (L.)tabacum2 L.
Nicotiana Salt
UV-Bstress ROS accumulation, DNA fragmentation, [40]
[43]
cv. Bright Yellow UV-B dysfunction
protease activity, mitochondrial [43]
Heynh.cv. Bright Yellow 2 Changes in cell anddysfunction
nucleus morphology, DNA
Pinus pinaster Ait. Sugar and phosphate depletion
mitochondrial dysfunction [44]
Changes in cell and nucleus
fragmentation, morphology, DNA
DNA laddering
Pinus pinaster Ait. Sugar and phosphate depletion 2+ [44]
Changes in ROS
cell accumulation,
fragmentation,
and nucleus DNA Ca influx,
laddering
morphology, DNA
Arabidopsis thaliana (L.)
Arabidopsis thaliana Ozone
polyethylene glycol, mannose, H2O2, Changes in cellchanges
and nucleus in
fragmentation, specific gene activation. cell and
morphology, DNA
* Cell plasma [41]
Heynh. [45]
Arabidopsis
(L.) Heynh.thaliana polyethylene glycol, *mannose, H2O2,
ethylene fragmentation,
membrane nucleus
specific gene
permeabilization, morphology
activation. * Cell plasma
ROS overproduction, [45]
(L.) Heynh. ethylene * membrane permeabilization,
severe ROS overproduction,
oxidative stress
ROS accumulation, DNA
* Main characteristics of PCD induced by severe oxidative stress
ethylene.
Vitis vinifera L. Darkness (in senescent cultures) fragmentation, caspase-3-like protease [42]
* Main characteristics of PCD induced by ethylene.
For example, cadmium is a potent activity,
inducermitochondrial
of PCD in plants dysfunction
and in tobacco BY-2-
Forcells;
cultured example, cadmium
this process is a potent
involves inducer
alterations in of PCD
cell and in plantsmorphology
nucleus and in tobaccoandBY-2-
ap-
ROS accumulation,
Nicotiana tabacum L. cv. cultured
pearance cells;
of this
autophagicprocess
bodiesinvolves
[31]. In alterations
the same in cell and
experimental nucleus morphology
material, aluminum and
oxideap-
UV-B DNA fragmentation, [43]
Bright Yellow 2 pearance of autophagic bodies [31]. In the
nanoparticles induce a PCD form closely connected same experimental
to loss of material, aluminum
mitochondrial potential, en- oxide
mitochondrial dysfunction
nanoparticles
hancement induce a PCD
of caspase-like form closely
activity, and DNA connected to loss of[32].
fragmentation mitochondrial potential,
In Viola tricolor L.-cul-en-
hancement
tured of caspase-like
cells, zinc and lead ionsactivity,
stimulate Changes
and aDNA in showing
cell and[32].
PCD fragmentation
form nucleus
DNAInfragmentation
Viola tricolor L.-cul-
and
Pinus pinaster Ait. tured cells,
Sugar activation
and of zinc
phosphate and leadand
depletion
caspase-like ionspapain-like
stimulate acysteine
PCD form
morphology, DNA showing
proteases DNA fragmentation
fragmentation,
[33]. Interestingly, and
the[44]
in-
activation of caspase-like and papain-like cysteine
DNA proteases
laddering [33]. Interestingly,
doleamine melatonin protects tobacco BY-2-cultured cells from lead stress by inhibiting the in-
doleamine melatonin protects tobacco BY-2-cultured cells from lead stress by inhibiting
Changes in cell and nucleus
morphology, DNA fragmentation,
Arabidopsis thaliana (L.) polyethylene glycol, mannose, H2 O2 ,
specific gene activation. * Cell plasma [45]
Heynh. ethylene *
membrane permeabilization, ROS
overproduction, severe oxidative stress
* Main characteristics of PCD induced by ethylene.Int. J. Mol. Sci. 2021, 22, 2166 8 of 12
For example, cadmium is a potent inducer of PCD in plants and in tobacco BY-
2-cultured cells; this process involves alterations in cell and nucleus morphology and
appearance of autophagic bodies [31]. In the same experimental material, aluminum oxide
nanoparticles induce a PCD form closely connected to loss of mitochondrial potential, en-
hancement of caspase-like activity, and DNA fragmentation [32]. In Viola tricolor L.-cultured
cells, zinc and lead ions stimulate a PCD form showing DNA fragmentation and activation
of caspase-like and papain-like cysteine proteases [33]. Interestingly, the indoleamine
melatonin protects tobacco BY-2-cultured cells from lead stress by inhibiting cytochrome
c release, thereby preventing the activation of the cascade of processes leading to cell
death [46]. Other important environmental pollutants able to induce PCD in cultured plant
cells are aromatic compounds. In fact, fluoranthene causes DNA fragmentation and oxida-
tive stress in tobacco BY-2 suspension cultures [34]. Rose Bengal dye in Arabidopsis thaliana
cell suspension cultures requires functional chloroplasts to activate a PCD process showing
ROS accumulation and specific gene activation [35], and the herbicide dinitro-o-cresol in-
duces DNA fragmentation, activation of caspase-3-like proteins, and release of cytochrome
c from mitochondria in soybean (Glycine max) suspension cell cultures [36]. Other chemicals
able to induce PCD are 1-butanol, which in Populus euphratica cell cultures causes shrinkage
of the cytoplasm, DNA fragmentation, condensed or stretched chromatin, and the activa-
tion of caspase-3-like proteases [37] and ATP, which when externally added to the same
cell cultures causes elevation of cytosolic Ca2+ levels, ROS accumulation, and cytochrome c
release [38]. As far as environmental conditions are concerned, heat stress (HS) is a potent
inducer of PCD in plants, where it causes important yield losses. HS study in cultured cells
has permitted to elucidate some aspects of its induction, thus helping in the reduction of
losses. For example, in tobacco BY-2-cultured cells, HS induces PCD, showing apoptotic
features such as cytoplasmic shrinkage, DNA fragmentation, ROS accumulation, activation
of caspase-3-like proteases, and induction of defense-related genes [39,47]. Some of these ef-
fects of HS are prevented by selenium [47] and depend on peroxynitrite accumulation [48],
thus sustaining the fundamental role of oxidative stress in the induction of HS-dependent
PCD. This view is also sustained by the analysis of the soluble proteome of tobacco cells
subjected to HS and by custom microarray analysis of gene expression during PCD of
Arabidopsis thaliana-cultured cells. Both these molecular investigations show the induction
of genes related to oxidative stress resistance [49,50]. Another environmental condition
that is able to induce PCD is salinity. Interestingly, the comparison of the responses to salt
stress of suspension-cultured cells from the halophyte Cakile maritima and the glycophyte
Arabidopsis thaliana shows that both species present similar dysfunction of mitochondria
and caspase-3-like activation but the salt-tolerant C. maritima can better resist to stress due
to a higher ascorbate pool able to mitigate the oxidative stress generated in response to
NaCl [40]. O3 exposure also induces PCD dependent on ROS generation in cell suspensions
of Arabidopsis thaliana [41]. Light also seems to be an important environmental factor able
to regulate PCD. Darkness enhances cell death but flavonoids and darkness lower PCD
during senescence of Vitis vinifera cell suspensions [42], pointing out the complexity of PCD
regulation in plants. In tobacco BY 2-cultured cells, UV-B overexposure induces a PCD form
showing typical apoptotic morphological features such as cell shrinkage, condensation of
chromatin in perinuclear areas, and formation of micronuclei [43]. The nutritional aspect
is also important. In fact, simultaneous depletion of sugar and phosphate is associated
with PCD, showing nuclear DNA degradation in suspension cultures of maritime pine
(Pinus pinaster Ait.) [44].
Very interesting results have been obtained from experiments performed in a cell
cycle-synchronized Arabidopsis thaliana cell suspension culture treated with four physiolog-
ical stressors (polyethylene glycol, mannose, H2 O2 , ethylene) in the late G2 phase. In these
cultures, depending on the cell death inducer, there are significant differences in the ap-
pearance of specific PCD hallmarks. In fact, polyethylene glycol, mannose, and H2 O2 cause
DNA fragmentation and cell permeability to vital stains, and produce corpse morphology
corresponding to apoptotic-like PCD. Instead, ethylene (a plant hormone associated withInt. J. Mol. Sci. 2021, 22, 2166 9 of 12
senescence) causes permeability of cells to vital stains without concomitant nuclear DNA
fragmentation and cytoplasmic retraction but with very high ROS production, leading to
severe oxidative stress [45]. Similarly, in tobacco BY 2-cultured cells, zinc oxide nanopar-
ticles cause cell death depending on oxidative stress and lipid peroxidation [51], and in
grapevine suspension cell cultures, different concentrations of silver ions cause cell death
with different characteristics [52]. Thus, depending on the genotype/species and level of
stress, the same factors may cause different responses. Low stress levels permit the repair
of cell damage, moderate stress levels may induce PCD, and uncontrollable stress levels
Int. J. Mol. Sci. 2021, 22, 2166 potentially lead to accidental cell death (necrosis, see also Section 5). This is particularly
10 of 13
evident with abiotic stressors such as heavy metals and externally added compounds such
as plant hormones and H2 O2 (original articles cited in [53]).
5. Future Perspectives and Conclusions
5. Future Perspectives and Conclusions
The use of cultured cells, a simple and controllable system valuable for physiological
The use of cultured cells, a simple and controllable system valuable for physiological
studies because it minimizes variability and facilitates the analysis of cellular features,
studies because it minimizes variability and facilitates the analysis of cellular features,
permits theinvestigation
permits the investigationofofsome
some aspects
aspects of the
of the induction
induction and and progression
progression process
process of
of plant
plant
PCD. In particular, the possibility to evaluate the effects of different inducers in a stable anda
PCD. In particular, the possibility to evaluate the effects of different inducers in
stable and reproducible
reproducible manner made manner made it
it possible topossible to investigate
investigate the existencetheofexistence
multipleofpathways
multiple
pathways to perform PCD in plants. For example, the comparison
to perform PCD in plants. For example, the comparison among the effect of tunicamycin,among the effect of
tunicamycin,
brefeldin A and brefeldin A and
fusicoccin andfusicoccin
the lack ofand the lackwhen
additivity of additivity when these
these inducers inducers
are furnished
are furnished in combination to Acer pseudoplatanus cells, permitted to
in combination to Acer pseudoplatanus cells, permitted to hypothesize for these inducers hypothesize for
these inducers the existence of the same or largely coincident pathways
the existence of the same or largely coincident pathways involving mitochondria and involving mito-
chondria
endoplasmicand reticulum
endoplasmic reticulum
to induce PCD to[54].
induceOnPCD [54]. hand,
the other On thethe
other hand, thebetween
discrepancy discrep-
ancy between the number of dead cells and the number of cells showing
the number of dead cells and the number of cells showing specific DNA fragmentation specific DNA
fragmentation suggestsofthe
suggests the presence presence
different of different
types of PCD types of PCD
in these in these
cultures [54].cultures [54]. This
This permits the
permits
hypothesisthe of
hypothesis of the
the existence of existence
at least twoof at leasttotwo
ways die ways to die plant
in cultured in cultured plant cells
cells (Scheme 1):
(Scheme 1): apoptotic-like
apoptotic-like PCD and necrosis.PCD and necrosis.
Scheme 1. Possible ways to die in plant-cultured cells.
The apoptotic-like
The apoptotic-likePCD PCDisisprobably
probably thethe most
most present
present in cultured
in cultured cellscells and occurs
and occurs dur-
during
ing developmental
developmental processes
processes and and in response
in response to pathogen
to pathogen attackattack (Tables
(Tables 1 and1 2).
andThe
2).
The biotic and abiotic cell death inducers stimulate ROS and RNS production
biotic and abiotic cell death inducers stimulate ROS and RNS production and changes in and changes
in cytosolic 2+ levels. These second messengers induce activation of specific genes,
cytosolic Ca2+Ca
levels. These second messengers induce activation of specific genes, leading
leading
to DNA to DNA fragmentation
fragmentation as well as asmitochondrial
well as mitochondrial dysfunction
dysfunction with activation
with activation of
of caspase-
caspase-like proteases, finally resulting in apoptotic-like PCD. The necrotic death
like proteases, finally resulting in apoptotic-like PCD. The necrotic death involves swell- involves
swelling
ing of theof the protoplast
protoplast andofloss
and loss the of the integrity
integrity of the plasma
of the plasma membrane membrane
and occursand during
occurs
during the response to severe insults that cause an oxidative uncontrolled
the response to severe insults that cause an oxidative uncontrolled shock. shock.
The absence
The absence ofof cellular
cellulardifferentiation
differentiationininmany
manysuspension
suspension cultures
culturesis aismain
a mainadvan-
ad-
vantage when the basal responses of cells during the induction and execution of cell death
tage when the basal responses of cells during the induction and execution of cell death
programs are investigated. However, it is possible to obtain suspension cultures not totally
programs are investigated. However, it is possible to obtain suspension cultures not to-
tally undifferentiated to study some developmental processes. In fact, the developmental
cell deaths that occur in culture are often more amenable to investigation than their in
vivo counterparts. A classic example was the use of the xylogenic Zinnia (Zinnia elegans)
cell culture as an efficient system for studies on xylogenesis, an example of the “vacuolarInt. J. Mol. Sci. 2021, 22, 2166 10 of 12
undifferentiated to study some developmental processes. In fact, the developmental cell
deaths that occur in culture are often more amenable to investigation than their in vivo
counterparts. A classic example was the use of the xylogenic Zinnia (Zinnia elegans) cell
culture as an efficient system for studies on xylogenesis, an example of the “vacuolar first”
form of PCD. The developmental program of xylem differentiation in planta is well pre-
served in this in vitro experimental model. This allows for easy identification of signaling
molecules and observations of the changes in the morphology of the cellular organelles [55].
The background provided by the xylogenesis research in Zinnia makes it possible to uti-
lize a similar approach to study, in a simplified model, cell cultures other fundamental
processes for the plant lifestyle such as embryogenesis, sex determination, or senescence.
In conclusion, the importance and complexity of the PCD process in plants needs
further investigation, and in this perspective, cell cultures appear a very useful tool.
Author Contributions: Conceptualization, M.M. and R.C.; funding acquisition, R.C.; investigation,
M.M.; writing—original draft, M.M.; writing—review and editing, R.C. All authors have read and
agreed to the published version of the manuscript.
Funding: This research is supported by the University of Milano-Bicocca, Fondo d’Ateneo per
la Ricerca.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
HS Heat stress
PCD Programmed cell death
RNS Reactive nitrogen species
ROS Reactive oxygen species
References
1. Jones, A.M. Programmed cell death in development and defense. Plant Physiol. 2001, 125, 94–97. [CrossRef] [PubMed]
2. Lam, E. Controlled cell death, plant survival and development. Nat. Rev. Mol. Cell Biol. 2004, 5, 305–315. [CrossRef]
3. Reape, T.J.; Molony, E.M.; McCabe, P.F. Programmed cell death in plants: Distinguishing between different modes. J. Exp. Bot.
2008, 59, 435–444. [CrossRef]
4. Van Aken, O.; Van Breusegem, F. License to kill: Mitochondria, chloroplast and cell death. Trends Plant Sci. 2015, 20, 754–766.
[CrossRef] [PubMed]
5. Van Doorn, W.G.; Beers, E.P.; Dangl, J.L.; E Franklin-Tong, V.; Gallois, P.; Hara-Nishimura, I.; Jones, A.M.; Kawai-Yamada, M.;
Lam, E.; Mundy, J.; et al. Morphological classification of plant cell deaths. Cell Death Differ. 2011, 18, 1241–1246. [CrossRef]
6. McCabe, P.F.; Leaver, C.J. Programmed cell death in cell cultures. Plant Mol. Biol. 2000, 44, 359–368. [CrossRef] [PubMed]
7. Crosti, P.; Malerba, M.; Bianchetti, R. Tunicamycin and Brefeldin A induce in plant cells a programmed cell death showing
apoptotic features. Protoplasma 2001, 216, 31–38. [CrossRef]
8. Malerba, M.; Cerana, R.; Crosti, P. Fusicoccin induces in plant cells a programmed cell death showing apoptotic features.
Protoplasma 2003, 222, 113–116. [CrossRef]
9. Duval, I.; Brochu, V.; Simard, M.; Beaulieu, C.; Beaudoin, N. Thaxtomin A induces programmed cell death in Arabidopsis thaliana
suspension-cultured cells. Planta 2005, 222, 820–831. [CrossRef]
10. Cheng, D.D.; Jia, Y.J.; Gao, H.Y.; Zhang, L.T.; Zhang, Z.S.; Xue, Z.C.; Meng, Q.W. Characterization of the programmed cell death
induced by metabolic products of Alternaria alternata in tobacco BY-2 cells. Physiol. Plant 2011, 141, 117–129. [CrossRef]
11. Jiao, J.; Sun, L.; Zhou, B.; Gao, Z.; Hao, Y.; Zhu, X.; Liang, Y. Hydrogen peroxide production and mitochondrial dysfunction
contribute to the fusaric acid-induced programmed cell death in tobacco cells. J. Plant Physiol. 2014, 171, 1197–1203. [CrossRef]
[PubMed]
12. Hirakawa, Y.; Nomura, T.; Hasezawa, S.; Higaki, T. Simplification of vacuole structure during plant cell death triggered by culture
filtrates of Erwinia carotovora. J. Integr. Plant Biol. 2015, 57, 127–135. [CrossRef] [PubMed]
13. Yekkou, A.; Tran, D.; Arbelet-Bonnin, D.; Briand, J.; Mathieu, F.; Lebrihi, A.; Errakhi, R.; Sabaou, N.; Bouteau, F. Early events
induced by the toxin deoxynivalenol lead to programmed cell death in Nicotiana tabacum cells. Plant Sci. 2015, 238, 148–157.
[CrossRef]
14. Townley, H.E.; McDonald, K.; Jenkins, G.I.; Knight, M.R.; Leaver, C.J. Ceramides induce programmed cell death in Arabidopsis
cells in a calcium-dependent manner. Biol. Chem. 2005, 386, 161–166. [CrossRef]
15. Iakimova, E.T.; Michaeli, R.; Woltering, E.J. Involvement of phospholipase D-related signal transduction in chemical-induced
programmed cell death in tomato cell cultures. Protoplasma 2013, 250, 1169–1183. [CrossRef]You can also read
