Plant Growth Regulation 37: 263285, 2002.

2002 Kluwer Academic Publishers. Printed in the Netherlands.

263

Concepts in plant stress physiology. Application to plant tissue cultures

T. Gaspar 1,*, T. Franck 1, B. Bisbis 1, C. Kevers 1, L. Jouve 2, J.F. Hausman 2 and J. Dommes 1
1

Department of Plant Biology, University of Lige Sart Tilman, B 22, B-4000 Lige, Belgium; 2CRP-Gabriel
Lippmann, 162A, avenue de la Faencerie, LU-1511 Luxembourg, GD, Luxembourg; *Author for
correspondence (e-mail: Th.Gaspar@ulg.ac.be)
Received 30 August 2001; accepted in revised form 12 January 2002

Key words: Acclimation, Adaptation, Hyperhydricity, Plant cancerous state, Plant resistance, Plant stress, Plant
tissue cultures, Reactive oxygen species, Somaclonal variation
Abstract
Because the term stress is used, most often subjectively, with various meanings, this paper first attempts to clarify
the physiological definition, and the appropriate terms as responses in different situations. The flexibility of normal metabolism allows the development of responses to environmental changes which fluctuate regularly and
predictably over daily and seasonal cycles. Thus every deviation of a factor from its optimum does not necessarily result in stress. Stress begins with a constraint or with highly unpredictable fluctuations imposed on regular metabolic patterns that cause bodily injury, disease, or aberrant physiology. Stress is the altered physiological
condition caused by factors that tend to alter an equilibrium. Strain is any physical and/or chemical change produced by a stress, i.e. every established condition, which forces a system away from its thermodynamic optimal
state. The paper secondly summarises the Strassers state-change concept which is precisely that suboptimality is
the driving force for acclimation (genotype level) or adaptation (population level) to stress. The paper continues
with the actual knowledge on the mechanisms of stress recognition and cell signalling. Briefly: plasma membranes are the sensors of environmental changes; phytohormones and second messengers are the transducers of
information from membranes to metabolism; carbon balance is the master integrator of plant response; betwixt
and between, some genes are expressed more strongly, whereas others are repressed. Reactive oxygen species
play key roles in up- and down-regulation of metabolism and structure. The paper shows finally that the above
concepts can be applied to plant tissue cultures where the accumulating physiological and genetical deviations
(from a normal plant behaviour) are related to the stressing conditions of the in vitro culture media and of the
confined environment. The hyperhydrated state of shoots and the cancerous state of cells, both induced under
conditions of stress in in vitro cultures, are identified and detailed, because they perfectly illustrate the stressinduced state-change concept. It is concluded that stress responses include either pathologies or adaptive advantages. Stress may thus contain both destructive and constructive elements: it is a selection factor as well as a
driving force for improved resistance and adaptive evolution.
Abbreviations: AA, DHA ascorbic acid, dehydroascorbic acid, CIT citric acid, DAO diamine oxidase,
GABA -aminobutyric acid, GSSG, GSH oxidized glutathione, reduced glutathione, HNE 4-hydroxynonenal, HS hyperhydric shoots, HS-Ps heat-shock (related) proteins, KG -cetoglutarate, LEA-Ps late embryogenesis abundant-proteins, MAL malate, MDE malondialdehyde, MDH malate dehydrogenase, NS
normal shoots, OAA oxaloacetic acid, OPP oxidative pentose phosphate pathway, PAs polyamines, PAO
polyamine oxidase, PR-Ps pathogenesis-related proteins, PS photosystem, ROS reactive oxygen species,
PEP(C) phosphoenolpyruvate (carboxylase), SAR systematic acquired resistance, SOD superoxide dismutase, SSA succinate semialdehyde, SUC succinate.

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Introduction
Environmental stresses represent the factors which
most limit agricultural productivity worldwide. These
stresses not only have an impact on current crop species, but they are also significant barriers to the introduction of crop plants into areas that are not currently
being used for agriculture. Stresses associated with
temperature, salinity and drought, single or in combination, are likely to enhance the severity of problems
to which plants will be exposed in the coming decades (Duncan 2000; Cherry et al. 2000). Major efforts to breed for traits that confer tolerance of
drought, cold, heat, nutrient, and salinity stress are
already made each year throughout the world. An understanding of the mechanisms that regulate form and
function, and the significance of those processes to
plant physiology, ecology and agriculture must include knowledge of plant stress physiology.
Metabolic, anatomical and morphological responses to stress are some of the primary processes
of microevolution by natural selection. Therefore, one
of the major forces that shapes the structure and function of plants is environmental stress. The significance of adaptive responses to environmental stress
also is highlighted by the many cases of convergent
evolution in plants. Similarities in form and function
among phylogenetically unrelated plants are often a
consequence of environmentally driven coevolution
(Nilsen and Orcutt 1996).
Because the term stress is used, most often subjectively, with various meanings, the first aim of the
present paper is to clarify the physiological definition,
and the appropriate terms as responses in different
situations. The paper will secondly summarise the
Strassers state-change concept where stress, in the
sense of the physiological state, is the condition
caused by factors that tend to alter an equilibrum. The
paper shows finally that this concept can be applied
to situations encountered during plant tissue cultures.

large seasonal variations in temperature (including

freezing), the aerial parts of the plant may face temperature variations of tens of degrees centigrade in a
single day and smaller temperature changes in a matter of minutes.
Many of the environmental factors, which fluctuate, are associated intimately with metabolic processes. Variation in light, which supplies energy for
photosynthesis, has immediate effects on metabolism,
while water deficit decreases stomatal conductance
and therefore limits carbon dioxide supply and
changes the balance between photosynthesis and photorespiration. The source (nitrate vs. ammonium) and
concentration of nitrogen influences the location (root
or shoot) and rate of nitrogen assimilation into amino
acids and therefore requires a dynamic balance between photosynthesis, carbon partitioning and nitrogen assimilation (Smirnoff 1995). Thus the environment around plants fluctuates regularly and
predictably under daily and seasonal cycles. The flexibility of their metabolism allows plants to deal with
their constantly fluctuating environment. Co-ordination of plant metabolic activity with the surrounding
environmental factors (i.e. abiotics and biotics: temperature, water, light, UV radiation, mineral nutrient,
oxygen supply, bacteria, myccorhizal fungi...) has
major effects on plant growth.
Stress arising from unpredictable environmental
variations causes partitioning changes in the plant that
are plastic, but the degree of response is under intraand inter-species genetic control. When the environmental stress is relatively predictable over the life
span of a plant, responses to the stresses become genetically fixed in the plant, leading to evolution of
ecotypes or landraces. Consequently stress responses
will govern the level of tolerance, ultimately affecting productivity and temporal stability to less than
optimal habitats (Duncan 2000).

Towards a stress definition

Metabolic flexibility in relation to the
environment
Plants, particularly in temperate climates, live in an
environment that changes, not only seasonally, but
also from one moment to the next. These rapid environmental changes largely involve fluctuations in
light and temperature. Such plants grow over a wide
range of temperatures and, apart from encountering

Though the term stress has been defined exactly in

mechanics, in the case of biology it has been given
widely different meanings. Probably due to an extension of the physical meaning, many of these definitions converge in attributing stress to any environmental factor unfavourable for the living organism
under consideration (Levitt 1980). In accordance, the
ability of the organism to survive the unfavourable
factor has been called stress resistance.

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Table 1. Some of the sources of environmental stress in plant
(Nilsen and Orcutt 1996).
Physical

Chemical

Biotic

Drought
Temperature
Radiation
Flooding
Wind
Magnetic field

Air pollution
Heavy metals
Pesticides
Toxins
Soil pH
Salinity

Competition
Allelopathy
Herbivory
Diseases
Pathogenic fungi
Viruses

Every change of an environmental factor influences plant growth and development. Every deviation
of a factor from its optimum is not necessarily a stress
for a flexible plant acclimatised to its environment.
Stress begins with a constraint (biotic or abiotic) (Table 1) or with highly unpredictable fluctuations imposed on the regular metabolic pattern, that cause
bodily tension. Stress, in the sense of a stressor or
stress inducer (Cassells and Curry 2001), is an unusual factor or a usual factor of the biotic and abiotic
environment modified in such a way (excess or deficit) that it has the capability of causing bodily injury,
disease, or aberrant physiology. The disease is considered as a condition of the living (animal or plant)
body or one of its parts that impairs the performance
of a vital function, or functions, as a response to environmental factors or inherent genetic defects.
Stress, in the sense of the physiological state, is the
condition caused by factors that tend to alter an equilibrium (Nilsen and Orcutt 1996).
Because plants are confined to the place where
they grow, they have a limited capacity to avoid unpredictable unfavourable changes in their environment (confrontation with extremes of temperature,
water shortage, insufficient or excessive light or mineral nutrients, attack by pathogenic bacteria, fungi,
viruses and viroids) (Table 1). They have developed
ingenious molecular strategies to defend themselves
against such biotic and abiotic stresses, most often
combined with an alteration of growth and developmental patterns. This explains why the concept of
stress is intimately associated with the external conditions that adversely affect growth, development or
productivity (Lutts and Kinet 1998).
Other authors define stress as changes in physiology that occur when species are exposed to extraordinary unfavourable conditions that need not represent a threat to life but will induce an alarm response
(Larcher 1980). Alarm responses are defensive or

adaptive responses to the stimulus. This definition can

be difficult to use. For example, if plants are placed
in the sun and water is withheld, leaves may wilt.
Wilting could be detrimental to the plant in the short
term because carbon gain is inhibited. But wilting
may be critical for survival in the long run because
leaf temperature may be kept low enough to avoid
permanent damage (less light energy is absorbed by a
wilted leaf). Therefore, wilting is both a detrimental
and an advantageous response to low water availability and high light (Nilsen and Orcutt 1996). This gray
distinction between adaptive mechanism and detrimental effect on physiology prompted the consideration of the additional term strain (Levitt 1980).
Differentiation between stress and strain comes
from applying the engineering meaning of these terms
to biological systems. The biophysical definition of
stress is that it is the applied force divided by the area
of the force, or pressure. The term stress, in a plant
physiological sense, is therefore reflective of the
amount of environmental pressure for change that is
placed on an organisms physiology. Strain is defined
as the proportional change in a substance as a consequence of stress. Strain can be characterised as a
physiological change that occurs in response to environmental stress that does not necessarily result in
significant reduction of growth or reproduction (Levitt 1982).
According to Strasser (1988), strain is any physical and/or chemical change produced by a stress (Figure 2), which is every established condition that
forces a system away from its thermodynamically optimal state. The state is called optimal when the biological system is in full harmony with its environment. Harmony of a biological system with its
environment is achieved when the system does not
tend to change any activity or conformation whatsoever.
To summarise, although the term stress may be
used for indicating the STRESSOR agent (that is the
unusual factor, or a usual factor modified in such a
way, excess or deficit, that it has the capability of
causing bodily injury or disease), or for the alarm
RESPONSE to extraordinarily unfavourable conditions (for instance wilting), it primarily concerns the
altered PHYSIOLOGICAL STATE, impairing the
performance of a vital function.

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Figure 2. Strain as defined in the Strasser (1988) state-change concept (see Figure 1).

offers the possibility of analytical description and

quantification. In this concept, no environmental factor is considered a priori as unfavourable and the
plant does not have to resist, but it simply reacts.
As far as the system manages to adapt, which means
that the attraction point is within realistic limits
(Tsimilli-Michael et al. 1996), stress is not only harmless but, even more, constructive because it results in
improved resistance and adaptive evolution. However, if the adaptability of the system is overtaxed
then stress is destructive, leading to permanent damages or even to death (Figure 3). This Strassers stress
concept is in full agreement with Larchers definition:
Every organism experiences stress, although the way
in which it is expressed differs according to its level
of organisation. From the botanists point of view,
stress can be described as a state in which increasing
demands made upon a plant lead to an initial destabilisation of functions, followed by normalisation and
improved resistance. If the limits of tolerance are exceeded, permanent damage or even death may result.
Stress thus contains both destructive and constructive
elements: it is a selection factor as well as driving
force for improved resistance and adaptive evolution.

Physiological responses
The duration, severity, and rate at which a stress is
imposed all influence how a plant responds. Several

adverse conditions in combination may elicit a response differing from that for a single type of stress.
Features of the plant, including organ or tissue identity, development age, and genotype, also influence
plant response to stress (Bray et al. 2000). At specific
developmental stages, plants are either more or less
sensitive to particular stressors. The sensitivity stages
of development are called windows of sensitivity. A
response may be triggered directly by a stress, such
as drought, or may result from a stress-induced injury,
such as loss of membrane integrity. Some responses
clearly enable a plant to resist to stress, whereas the
functional role of others is not apparent (Figure 3).
Do somaclonal variations and mutations simply represent accidents? Failure to compensate for a severe
stress can result in somaclonal variation or mutation
(Cassells et al. (1997, 1999a, 1999b)), in complete
loss of organogenic totipotency, and ultimately in
plant or cell death, directly or following a neoplasic
progression (Lambe et al. 1997; Gaspar et al. 1998,
2000; Gaspar 1999).
Mechanisms that permit stress survival are termed
RESISTANCE mechanisms and can allow an organism to tolerate or avoid stress. Thus, physiological
responses to stressors can be divided into three possibilities. In one case, TOLERANCE, plants have
mechanisms that maintain high metabolic activity
(similar to that in the absence of stress) under mild
stress and reduced activity under severe stress. In
contrast, mechanisms of AVOIDANCE involve a reduction of metabolic activity, resulting in a dormant

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Figure 3. Plant responses to environmental stress in correspondence with stress and plant characteristics.

state, upon exposure to extreme stress (Osmond et al.

1987). Commonly, a plant species may have several
tolerance or avoidance mechanisms, or a combination
of both. For instance, drought stress may induce
drought tolerance that can be followed by desiccation
tolerance: in the later dormant state, the organism
can survive the dry state for long periods, i.e. years.
Notice that the ability to rehydrate without damage
can be considered as a part of the desiccation tolerance. The other issue is immediate or delayed DAMAGES, through somaclonal variation, mutation, neoplastic progression, ultimate death via necrosis and/or
apoptosis (Figure 3).
The intensity of stress (pressure to change exerted
by a stressor) is not easily quantified. Stress could
occur at a low level, creating conditions that are marginally non-optimal, with little effect expected. However, if this mild stress continues for a long time,
becoming chronic stress, the physiology of plants is
likely to be altered. In contrast, conditions could become difficult quickly, resulting in acute condition.
This shock pattern of stress is likely to induce significant changes in a short time frame. Toxicologists,
particularly those in the area of pollution studies,
have developed the concept of dose. Dose is defined
to be the magnitude of perturbation times the length
of time the stress is applied. It thus accounts for the
influence of both intensity and duration on physiolog-

ical performance. Stress can be dramatic when it is

applied for a short duration and high intensity, or
when it is applied for a long duration at low intensity.
Plant responses to chronic stress and acute stress may
be very different even though the dose is the same.
In plant stress physiology an important distinction
must be made between ultimate (ADAPTATION) and
proximal (ACCLIMATION) plant responses. Adaptation occurs by various mechanisms at the genetic
level in populations over many generations. Microevolutionary processes change gene frequencies of a
population over time. In a stressful environment, it is
logical to assume that specific genotypes with appropriate gene combinations (those that confer the ability to survive and reproduce) are dominant in the population. Those particularly favourable gene
combinations in plants that inhabit stressful environments are called adaptations.
Populations that have adapted through evolutionary processes acting at the genetic level to a particular climatic regime are by no means static systems.
On the contrary, plants have an incredible ability to
adjust physiological and structural attributes on the
scale of seconds or seasons within a single genotype:
this is acclimation. In other words, during acclimation, an organism alters its homeostasis, its steadystate physiology, to accommodate (further) shifts in
its external environment. For instance, prolonged ex-

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vive heat stress. These include limiting or avoiding
direct absorption of solar radiation, dissipation of excess absorbed radiation, and physiological mechanisms that counteract the effects of heat stress on metabolism. All three strategies of tolerance are equally
important for survival. These strategies of tolerance
have arisen through the evolution of specific developments in plant morphology, anatomy and physiology (Luthe et al. 2000)
What is said above also means that, contrary to the
general opinion, stresses must not be automatically
associated with adverse detrimental effects. That is
like the breathless and fatiguing training of a sportsman that prepares him for a greater performance
(Strasser, personal comment). Stress responses thus
include pathologies and adaptive advantages, either
both successively or together.
Figure 4. Acclimation and hypersensitive reaction as possible intermediary steps in resistance and SAR (systemically acquired resistance against pathogens) to abiotic and biotic stresses, respectively.

Stress recognition and cell signalling

posure of chilling resistant plants to cold results in the

adjustment of plant growth and metabolism to low
temperature conditions and in the increased resistance
of tissues to freezing temperatures (Jouve et al. 2000).
Freezing resistance is the ability of plants to survive
formation of ice in tissues. Systematic acquired resistance (SAR) against pathogens is another form of acclimation (Figure 4).
On a long-term scale, acclimation is enhanced in
plants because of the modular nature of metabolism
and growth. Plant parts can be abscised and regrown
in a new morphology or anatomy, specific organs can
be enhanced by increasing their numbers or size. On
a short-term basis (i.e. seconds or minutes), protein
populations can ebb and wane, growth regulators can
be released or activated, or transcription and translation can be regulated up or down.
Acclimation is a phenotypic response to different
combinations of environmental characteristics. Phenotypic plasticity is an index of the amount of acclimation that is possible within one genotype (Nilsen
and Orcutt 1996).
Thus, adaptation at the population level, or acclimation at the level of the individual plant, occur
through a combination of behavioural, morphological, anatomical, physiological and biochemical processes, which depend on processes at the molecular
level. As another example, plants have evolved varied and multiple mechanisms that allow them to sur-

A stress response, with changes in metabolism and

development, is initiated when the plant recognises a
stress at the cellular level. Even if disintegrated cell
walls can be indirect sources of elicitors, intact, disrupted or damaged (plasma) membranes are the sensors of environmental change. Phytohormones and
second messengers (Ca 2+ for instance) are the transducers of information from membranes to metabolism. Carbon balance is the master integrator of plant
response (Nilsen and Orcutt 1996). Betwixt and between, some genes are expressed more strongly,
whereas others are repressed. The protein products of
stress-induced genes often accumulate in response to
unfavourable conditions (Figure 5). Although most
studies have focused on transcriptional activation of
gene expression, growing evidence suggests that the
accumulation of gene products is also influenced by
posttranscriptional regulatory mechanisms that increase the amounts of specific mRNAs, enhance
translation, stabilise proteins, alter protein activity, or
some combinations of these (Bray et al. 2000). After
stress recognition, the signal is communicated within
cells and throughout the plants. All parameters that
regulate cell growth must act through a small number
of physical processes. These are cell wall rheology,
water transport and solute transport. Auxins, gibberellins, abscisic acid, cytokinins and ethylene have all
been implicated in influencing one or more of these
processes (Tomos 1990).

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Figure 5. Cell signalling mediators in metabolic and structural adaptation to stress. Membranes are the sensors; phytohormones and second
messengers are the transducers; carbon balance is the master integrator; in between, some genes are expressed, others are repressed.

It is important to notice that plant responses to different stresses may use common ways. It is also true
that apparently different stresses such as drought,
heat, cold and salt may result in the same ultimate
stress, water deficit. On the other hand, mostly depending on the plant genera or families, a diversity of
responses to the same stress can exist because the
same function can be fulfilled by different compounds. For example, starch and fructan may be
equally effective as carbon storage compounds, while
sucrose and polyols such as mannitol and sorbitol
could be equally effective as carbon transport compounds in the phloem.
Components of the signalling pathways thus are
mostly similar to those implicated in other signalling
cascades in eucaryotes, and include reversible protein
phosphorylation steps, calcium/calmodulin-regulated
events, and production of active oxygen species (see
below) (Leon et al. 2001).
The interaction between different factors can be
another difficulty in the understanding of plant responses. For example, low air humidity creates an
environment that enhances the potential for signifi-

cant plant water loss. At the same time, regulating

water loss through stomatal closure causes a reduction in carbon dioxide (CO 2) diffusion that limits
growth. High air humidity, on the other hand, promotes environmental conditions favourable for disease development (Danneberger 2000).
The different possible signalling pathways, as well
as consequences of altered gene expression that alter
biochemical reaction downstream of stress sensing,
are discussed in several recent books and reviews
(Basra 1994; Smirnoff 1995; Nilsen and Orcutt 1996;
Asard et al. 1998; Lutts and Kinet 1998; Bray et al.
2000; Hasegawa et al. 2000; Wilkinson 2000; Zhu
2001).
Several imaging techniques have been used to detect the early signs of stress by monitoring changes
in water status, photosynthetic efficiency, accumulation of secondary metabolites or structural modifications. Thermography, reflectance detection and fluorescence imaging are currently the most highly
evolved of these techniques (Cassells et al. 1999a;
Chaerle and Van Der Straeten 2000).