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The Plant Journal - 2006 - Verslues - Methods and Concepts in Quantifying Resistance To Drought Salt and Freezing Abiotic

Methods and concepts in quantifyin

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The Plant Journal - 2006 - Verslues - Methods and Concepts in Quantifying Resistance To Drought Salt and Freezing Abiotic

Methods and concepts in quantifyin

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The Plant Journal (2006) 45, 523–539 doi: 10.1111/j.1365-313X.2005.02593.

TECHNIQUES FOR MOLECULAR ANALYSIS

Methods and concepts in quantifying resistance to drought,

salt and freezing, abiotic stresses that affect plant water
status
Paul E. Verslues, Manu Agarwal, Surekha Katiyar-Agarwal, Jianhua Zhu and Jian-Kang Zhu*
Institute for Integrative Genome Biology and Department of Botany and Plant Sciences, University of California, Riverside,
CA 92521, USA

Received 23 May 2005; revised 4 August 2005; accepted 6 September 2005.

*
For correspondence (fax 951 827 7115; e-mail [email protected]).

Summary
The abiotic stresses of drought, salinity and freezing are linked by the fact that they all decrease the availability
of water to plant cells. This decreased availability of water is quantified as a decrease in water potential. Plants
resist low water potential and related stresses by modifying water uptake and loss to avoid low water
potential, accumulating solutes and modifying the properties of cell walls to avoid the dehydration induced by
low water potential and using protective proteins and mechanisms to tolerate reduced water content by
preventing or repairing cell damage. Salt stress also alters plant ion homeostasis, and under many conditions
this may be the predominant factor affecting plant performance. Our emphasis is on experiments that quantify
resistance to realistic and reproducible low water potential (drought), salt and freezing stresses while being
suitable for genetic studies where a large number of lines must be analyzed. Detailed protocols for the use of
polyethylene glycol-infused agar plates to impose low water potential stress, assay of salt tolerance based on
root elongation, quantification of freezing tolerance and the use of electrolyte leakage experiments to quantify
cellular damage induced by freezing and low water potential are also presented.

Keywords: Arabidopsis, stress quantification.

Introduction
Abiotic stress limits crop productivity (Araus et al., 2002; stress), excess light (especially when photosynthesis is
Boyer, 1982), and plays a major role in determining the restricted) or increased hardness of the soil that restricts root
distribution of plant species across different types of envi- growth.
ronments. Abiotic stress and its effects on plants in both Several abiotic stresses are united by the fact that at least
natural and agricultural settings is a topic that is receiving part of their detrimental effect on plant performance is
increasing attention because of the potential impacts of caused by disruption of plant water status. This can occur
climate change on rainfall patterns and temperature through decreased availability of water in the environment
extremes, salinization of agricultural lands by irrigation, and during drought, altered ion content and water uptake caused
the overall need to maintain or increase agricultural pro- by salinity or cellular dehydration caused by formation of
ductivity on marginal lands. In the field, a plant may extracellular ice during freezing stress. Consequently, this
experience several distinct abiotic stresses either concur- paper focuses on these three stress factors: drought, salinity
rently or at different times through the growing season and freezing. In designing laboratory experiments to study
(Tester and Bacic, 2005). Some common examples of the plant responses to these stresses, the method used to
abiotic stresses a plant may encounter include a decreased impose the stress, the severity and duration of the stress, the
availability of water, extremes of temperature including parameters to be measured and how the observed
freezing, decreased availability of essential nutrients from responses of the plant fit into an overall strategy for resisting
the soil (or conversely the build-up of toxic ions during salt the stress are all important considerations. Evaluating the

ª 2006 Blackwell Publishing Ltd 523

1365313x, 2006, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2005.02593.x by Cochrane Mexico, Wiley Online Library on [18/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
524 Paul E. Verslues et al.

stress responses of mutants and transgenic plants is often more difficult for the plant to take up water, and this in
the most challenging type of experiment because the turn elicits a range of responses that allow the plant to
objective is to evaluate whether the plant’s overall perform- avoid water loss, allow water uptake to continue at re-
ance under stress has been altered. This is a much broader duced ww or allow the plant to tolerate a reduced tissue
question than the measurement of more narrowly defined water content. An overall picture of these responses must
parameters such as changes in gene expression or metabo- include changes in water fluxes and water relations at the
lite levels. Genetic studies can also be challenging because whole plant and the cellular levels.
the number of lines to be tested can demand a relatively high
level of throughput, thus constraining the type of experi-
Avoidance and tolerance of low ww
ments that are feasible.
In the case of crop plants, it is ultimately the yield of To understand the responses of plants to low ww at the level
genetically altered plants under specific field conditions that of the organism and cell it is useful to consider the stress
will determine whether or not a specific gene or metabolic or avoidance/stress tolerance terminology proposed by Levitt
signaling pathway is of technological importance. The (1972), a modified version of which is presented in Figure 1.
challenge of abiotic stress research is to bridge the gap In most cases, the plant’s first response is to avoid low ww.
between such agronomic or ecophysiological experiments Tissue ww and water content are maintained close to the
and the basic research in Arabidopsis and other model unstressed level by increasing water uptake or limiting water
organisms that is elucidating the molecular mechanisms by loss such that the rates of water loss and water uptake
which plants sense and respond to abiotic stress. It is in this remain balanced. Such a balance is achieved in the short
gap that the focus of this paper lies. Our goal is to discuss term mainly by stomatal closure. In the longer term, changes
relevant ideas and provide examples that will be of assist- in root and shoot growth, leading to an increased root/shoot
ance in designing experiments that are suitable for genetic ratio, tissue water storage capacity and cuticle thickness and
studies and rapid screening while still being relevant to water permeability are also of potential importance. Of
stress conditions in the real world. We first describe some these, changes in root growth to maximize water uptake are
basic principles of the responses of plants to altered water of the greatest importance for crop plants.
status. This background is then used to introduce experi- In the case of mild water stress or water stress of a limited
ments designed to examine responses to low water avail- duration, avoidance mechanisms by themselves can be
ability and to discuss the role of altered water status in sufficient to maintain plant performance (Kramer and Boyer,
salinity and freezing stress, and the methods used to impose 1995). Under such conditions, modifications such as
these stresses and evaluate plant resistance. Finally we increased root growth or decreased stomatal conductance
discuss some examples of the types of techniques useful in have the potential to increase crop productivity. The trade-
quantifying the extent of cellular and tissue damage caused off in this case is the lost photosynthesis caused by reduced
by abiotic stress treatments. stomatal CO2 uptake or a shift of resources into root growth
at the expense of photosynthetic and reproductive tissue.
Furthermore, these mechanisms for avoiding water loss do
Drought and low water potential
not themselves offer any protection from the effects of low
Although altered water status is a factor in a number of ww if the stress becomes more severe and the plant is no
abiotic stresses, it is of most obvious importance in longer able to maintain a balance between water uptake and
drought. Drought can be most simply defined as a period loss. In cases where low ww cannot be avoided by altering
of below normal precipitation that limits plant productivity water uptake and water loss, additional mechanisms
in a natural or agricultural system (Boyer, 1982; Kramer become important in maintaining plant function.
and Boyer, 1995). In the field, drought can cause a num-
ber of plant stresses including temperature, light and
Dehydration avoidance
nutrient stresses. However, the stress component that
defines drought is a decrease in the availability of soil When transpiration is minimized, as is likely to be the case
water. This decreased water availability can be quantified when stomata are closed because of stress, the ww of the
as a decrease in water potential (ww, Kramer and Boyer, plant will equilibrate with that of the water source (in most
1995). Mathematically, ww is the chemical potential of cases this is the soil ww). Thus, when soil water content and
water divided by the partial molar volume (Kramer and ww are low, ww of the plant tissue must also decrease, either
Boyer, 1995); thus, the free energy of water, as well as the through water loss or by adjustments made by the plant to
turgor of plant cells, can be expressed in units of pressure achieve a low ww while avoiding water loss. Such adjust-
and a straightforward assessment of the direction of wa- ments are termed ‘dehydration avoidance’ (Figure 1). The
ter movement in the soil/plant system can be made. De- main mechanisms of dehydration avoidance are accumula-
creased ww (decreased free energy of the water) makes it tion of solutes and cell wall hardening.

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

Figure 1. Conceptual diagram of the stress

tolerance/stress avoidance model of low-ww
responses.
In low-ww stress avoidance the plant balances
water uptake and water loss to avoid an effect of
the stress on tissue ww or water content (essen-
tially, the stress is kept outside the plant tissue). If
this cannot be achieved and the plant tissue does
experience low ww (the stress becomes internal-
ized to the plant tissue), stress responses occur
that maintain a high water content despite a
decreased ww (dehydration avoidance) or toler-
ate a reduced water content (dehydration toler-
ance). We use the term ‘stress resistance’ in
cases where it is not possible or not desirable to
refer to a more specific mechanism. The diagram
is based on the stress avoidance/stress tolerance
terminology of Levitt (1972).

Whether water will flow into or out of a plant cell is allowed the cell to maintain its original water content and
dependent on the ww gradient between the cell and its volume. In reality, of course, solute accumulation and water
surroundings. The ww of a walled cell, such as a plant cell, is loss can both occur in the same tissue and it is necessary to
governed by the equation: ww ¼ ws þ wp (Figure 2) where ws measure both change in volume (experimentally, change in
is the osmotic potential and wp is the pressure potential volume is often approximated as the change in relative
(turgor pressure). For a cell to take up water from the soil, or water content, Figure 2) and change in solute content to
other growth medium, it must have a lower ww than the calculate the extent of osmotic adjustment. This can be done
water source. An example of ww, ws and wp values which by calculating ws100 [the osmotic potential at 100% relative
could occur in a plant cell that is fully hydrated and exposed water content (Babu et al., 1999)] as shown for the examples
to a relatively high external ww is presented in Figure 2(a). in Figure 2(b).
At a given ww, a higher wp can be achieved by accumu- It is important that the solutes accumulated to prevent
lating solutes inside the cell, thus lowering ws. The accumu- water loss do not themselves interfere with cellular function.
lation of additional solutes in response to low ww is termed Thus, many plants accumulate one or more types of
osmotic adjustment (Zhang et al., 1999). Osmotic adjust- compatible solutes, such as proline or glycine betaine, in
ment refers to the active accumulation of additional solutes response to low ww, salinity, freezing and other abiotic
in response to low ww (after the effect of reduced water stresses that alter water status. These and other similar
content on the concentration of existing solutes has been solutes are termed compatible solutes because they can
factored out). Examples of plant cells exposed to low accumulate to high levels without interfering with metabo-
external ww are presented in Figure 2(b). The top cell in lism (Yancey et al., 1982) and may also have other protective
Figure 2(b) did not alter its solute content in response to properties. Osmotic adjustment and accumulation of com-
decreased external ww; the solute concentration inside the patible solutes can be an important factor in drought
cell did increase, but this was solely a result of decreased tolerance in the field (Kramer and Boyer, 1995; Morgan,
water content. In contrast, the middle cell in Figure 2(b) did 1984, 1991), and engineering of increased synthesis of
accumulate additional solutes in response to low ww and this compatible solutes is one approach that has been taken to

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

Figure 2. (a) Possible values of ww, ws (solute

content), wp (turgor), relative water content and
cell wall extensibility values for a plant cell
exposed to an external ww ()0.2 MPa) typical of
unstressed conditions.
(b) Examples of the alterations in values of water
relations, water content and cell wall extensibil-
ity after exposure to reduced external ww
()1.0 MPa) for three scenarios: no response
(top), solute accumulation (middle) and adjust-
ment of cell wall extensibility (bottom). Box:
definition of water relation terms.

increase abiotic stress tolerance in plants (Apse and Blum- loss of water causes little change in volume of the cell, but a
wald, 2002; Bohnert and Shen, 1999). The trade-off in this rapid decrease in turgor and ww that allows the cell to avoid
case is that increased accumulation of compatible solutes further water loss. This can be seen by comparing the top
can be energy and resource intensive for the plant, and, in cell in Figure 2(b), which has a relatively high e, with the
cases of severe stress where soil water content is largely bottom cell in Figure 2(b). Because of the high e, the bottom
depleted, may have only a small effect on water uptake cell is able to largely avoid dehydration even in the absence
(Kramer and Boyer, 1995). of solute accumulation. The trade-off of this strategy is that a
The properties of cell walls also play an important role in rigid cell wall and loss of turgor prevent any further
several abiotic stress responses, including dehydration expansion of the cell. Thus, increasing e to avoid water loss
avoidance. The deformability of the cell wall can be quan- is a strategy that is largely confined to non-growing tissues.
tified by the elastic modulus of the cell wall, e (Figure 2). Also, barring any increase in ww of the water source, solute
Simply stated, e is the pressure change required to cause a accumulation is still required for this cell to lower its ww and
unit change in cell volume (Kramer and Boyer, 1995; Murphy take up water.
and Ortega, 1995). When e is low, the cell wall deforms
readily; thus a loss of water will cause a large change in
Dehydration tolerance
volume but a small change in turgor because the cell wall
shrinks and continues to squeeze the cytoplasm. The high As low-ww stress becomes more severe, it becomes
turgor will cause ww to remain high, thus allowing further increasingly difficult for the plant to avoid dehydration and
water loss from the cell. In contrast when e is high, a small mechanisms to tolerate reduced water content become

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

important. The most dramatic examples of dehydration exclusively into one of the avoidance or tolerance categories
tolerance are ‘desiccation-tolerant’ plants that can recover shown in Figure 1. For example, accumulation of a com-
from a fully air-dried state (Oliver et al., 2000; Vicre et al., patible solute such as proline may play a role in dehydration
2004). When fully dehydrated, these plants are in a meta- avoidance by increasing the cellular solute content and thus
bolically dormant state that is in many ways similar to seed maintaining a higher water content. At the same time,
dormancy. Tolerance to severe dehydration is also a critical accumulation of proline has been proposed to play a role in
factor in freezing tolerance (see below). At the molecular dehydration tolerance by protecting protein and membrane
level, seed dormancy, freezing tolerance, the vegetative structure, regulating redox status or acting as a scavenger of
dormancy experienced by desiccation-tolerant plants and ROS (Hare et al., 1998; Hincha and Hagemann, 2004; Smir-
the dehydration responses in less tolerant species have noff and Cumbes, 1989; Verslues and Sharp, 1999). Likewise,
many similarities. However, most mesophytic plants (inclu- the dehydrin proteins may also act as ‘hydrophilins’, pro-
ding almost all crop plants) lack the ability to enter a dor- teins that bind water and thus could have a role in retaining
mant state to tolerate complete desiccation and thus cannot water (dehydration avoidance) in addition to a role in pro-
recover from a severe (approximately 50% or greater) de- tecting cellular structures (dehydration tolerance) (Close,
crease in water content. These plants instead attempt to 1997). Also, mechanisms that promote continued root
tolerate lesser degrees of water loss while maintaining growth at low ww, such as osmotic adjustment in the grow-
metabolic activity. ing region of the root (dehydration avoidance), may allow
Most of the dehydration tolerance mechanisms studied to roots to penetrate deeper into the soil and take up more
date function primarily to protect cellular structure from the water, thus contributing to avoidance of low ww.
effects of dehydration. Several types of protective proteins, It should also not be assumed that stress avoidance and
most notably dehydrins and other late-embryogenesis- tolerance occur in a linear progression in time after the
abundant (LEA) proteins, are well known to accumulate in stress begins or in a linear progression from responses
response to decreases in tissue water content either in initiated by mild stress to those initiated by severe stress. For
response to abiotic stress or during seed development example, some decrease in water content and turgor is likely
(Close, 1997). Although the function of many dehydrins and to be required to trigger accumulation of abscisic acid (ABA)
LEA proteins is not fully understood, at least part of their (Creelman and Zeevaart, 1985; Pierce and Rashke, 1980)
function is to act as chaperones that protect protein and which then causes stomatal closure to prevent further
membrane structure (Bravo et al., 2003; Hara et al., 2001). decrease in water content. Also, dehydration tolerance
Compatible solutes can also protect protein and membrane mechanisms such as accumulation of dehydrin and LEA
structure under dehydration (Hincha and Hagemann, 2004). protein may be initiated before significant dehydration
Another aspect of dehydration tolerance, and of tolerance to occurs as a way of preparing the plant for any further
other abiotic and biotic stresses, is the control of the level of decrease in water content. Rather than attempting to classify
reactive oxygen species (ROS) or limitation of the damage the various stress responses at a molecular level, a consid-
caused by ROS. The sources of ROS under stress, mecha- eration of tolerance and avoidance mechanisms is most
nisms of ROS detoxification and the role of ROS in stress useful in clarifying the appropriate types of experiments, the
signaling are all active areas of current research and have interpretation of the data and the terminology used to
been extensively studied and reviewed (Apel and Hirt, 2004; establish the role of a particular molecular event in the
op den Camp et al., 2003; Chen and Gallie, 2004; Corpas plant’s integrated response to low ww and other abiotic
et al., 2001; Foyer and Noctor, 2003; Hung et al., 2005; Jiang stresses.
and Zhang, 2003; Kwak et al., 2003; Laloi et al., 2004; Milla Given the overlapping functions of many low ww
et al., 2003; Moller, 2001; Mori and Schroeder, 2004; Pastori responses, it is perhaps not surprising that these responses
and Foyer, 2002; Shin and Schachtman, 2004). are controlled by a complex regulatory network. This
network responds to both external stimuli, such as loss of
turgor or reduced water content, and internal stimuli, such
An integrated response
as production of ROS, sugar sensing and various hormonal
The consideration of avoidance versus tolerance mecha- stimuli, that reflect the metabolic and developmental status
nisms provides a valuable framework for designing experi- of the plant (Verslues and Zhu, 2005). Although many of the
ments and interpreting the effects of low ww. Our molecular components involved in this regulation remain
understanding, however, of the molecular and cellular uncharacterized, ABA is well known to be a key regulatory
events that occur when plants are exposed to low ww has factor in controlling responses to many types of abiotic
increased greatly in the years since Levitt (1972) and others stress, including low ww. Abscisic acid accumulates in
proposed the ideas of avoidance and tolerance of low ww. response to abiotic stress and regulates the processes
With this increased understanding, it has become clear that involved in all of the aspects of the low-ww response
many of the molecular events initiated by low ww do not fit discussed above: ABA-regulated stomatal conductance and
ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539
1365313x, 2006, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2005.02593.x by Cochrane Mexico, Wiley Online Library on [18/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
528 Paul E. Verslues et al.

root growth (Schroeder et al., 2001; Sharp and LeNoble, mechanisms are important for all abiotic stresses but may
2002) are important in avoidance of low ww; ABA-induced play the dominant role in tolerance of the severe dehydra-
accumulation of compatible solutes can be crucial for tion caused by freezing.
dehydration avoidance (Ober and Sharp, 1994) and ABA- A number of genes have been suggested to be involved in
regulated synthesis of dehydrins and LEA proteins is different aspects of homeostasis or damage prevention
important for dehydration tolerance (Sivamani et al., 2000; under abiotic stress (Figure 3). Some examples are genes
Xu et al., 1996). Thus, at the level of the organism, it seems for the Naþ/Hþ antiporter SOS1 (Shi et al., 2000), the Naþ
that a main function of ABA is to coordinate the various influx transporter HKT1 (Rus et al., 2001, 2004) and the
aspects of low-ww response. A key aspect of understanding tonoplast Naþ/Hþ antiporter AtNHX1 (Apse and Blumwald,
low-ww response as a whole is a better understanding of the 2002), all of which are known to be important determinants of
upstream sensing and signaling that control ABA accumu- salt tolerance because of their role in ion homeostasis. Water/
lation and downstream signals that modulate the response osmotic homeostasis (dehydration avoidance) likely de-
to ABA (Verslues and Zhu, 2005; Zhu, 2002). The current pends on the action of genes for solute synthesis [such as
state of knowledge of perception of ABA, regulation of P5CS1 in Arabidopsis (Strizhov et al., 1997; Yoshiba et al.,
growth by ABA, ABA-dependent signal transduction and 1999)] and a number of channels and carriers for uptake and
ABA-regulated gene expression have been reviewed (Bray, compartmentalization of inorganic solutes, especially Kþ.
2002; Finkelstein et al., 2002; Sharp and LeNoble, 2002; Zhu, Aquaporins may also have a role in water and osmotic
2002). homeostasis by facilitating water movement; however, the
precise role of aquaporins in abiotic stress responses
remains undefined. One example of a protective protein for
The homeostasis and protection model
which a mechanism of action has been proposed is COR15.
The avoidance/tolerance model has been most commonly COR15 preserves membrane structure by preventing forma-
used to describe low-ww responses at the levels of the whole tion of the hexagonal phase and membrane fusion (Step-
plant and the cell. In addition to this model, molecular-level onkus et al., 1998). Regulatory proteins, for example ICE1
responses and responses to other abiotic stresses are often (Chinnusamy et al., 2003) and DREBs/CBFs (Shinozaki et al.,
discussed in terms of homeostasis and protection or dam- 2003), are critical for the induction of protective responses.
age repair (Zhu, 2001; Figure 3). These homeostatic mech- Overall, while general measurements of plant perform-
anisms include ion homeostasis, which is likely to be a ance such as growth and photosynthesis are applicable to
dominant factor in determining salt tolerance, and osmotic many types of abiotic stress experiments, consideration of
or water homeostasis, which is similar to the dehydration the mechanisms involved, either in the avoidance/tolerance
avoidance mechanisms discussed above and likely to be a terminology or in terms of homeostatic and protective
dominant factor in the low-ww response. Protection and re- mechanisms, will often suggest a more defined hypothesis
pair mechanisms are largely the same as the dehydration about the mechanisms by which a particular genetic change
tolerance mechanisms described above. These protective may affect the stress response. These hypotheses can then
be used to design more targeted experiments to quantify the
particular stress resistance mechanisms of greatest interest.
Some common experimental designs and the aspects of
stress avoidance, stress tolerance and homeostasis they
address are discussed in more detail below.

Experimental techniques for evaluating the low-ww

response

Here we describe some basic experimental designs

(Figure 4) that are suited to the evaluation of mutants and
transgenic plants: a number of lines can be tested in a fairly
high-throughput manner and relatively little specialized
Figure 3. Homeostasis and protection/damage repair model of the abiotic equipment or apparatus are required. Given this starting
stress response. point, Arabidopsis is used as the example plant. However,
Mechanisms of ion homeostasis and water/osmotic homeostasis attempt to
restore the cellular ion or water content to levels similar to those present the principles illustrated and, to a large extent, the experi-
under unstressed conditions. Protection and damage repair mechanisms mental techniques described, are applicable to other plants
attempt to prevent or repair cellular damage caused by altered ion or water as well. Particular attention is paid to consideration of which
content under stress. Some examples of genes involved in each class of
response are also shown. Arrows indicate interaction between these stress of the aspects of low-ww response discussed above is tested
response mechanisms. by each type of experiment.

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

Figure 4. Three types of experiments used to evaluate low-ww responses.

(a) Short-term avoidance of water loss using detached leaves or rosettes. The graph shows a typical result for decrease in fresh weight over time after detachment.
(b) Soil drying of pot-grown plants.
(c) Imposition of constant low ww under non-transpiring condition using PEG-infused agar plates. Preparation of PEG-infused plates is described in detail in Protocol
S1 of the Supplementary Material. The graph shows the typical pattern of ABA accumulation over time after transfer of 5- or 7-day-old seedlings from high ww
()0.25 MPa) to low ww ()1.2 MPa) using media without sugar. The acute response is the response from 0 to approximately 24 h after transfer. Recovery and longer-
term responses can be seen after 72 or 96 h or longer exposure to low ww.

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

water. This can lead to confusion if the severity of the stress is

Leaf water loss
not quantified by measuring leaf or soil ww or if steps are not
Perhaps the easiest experiment to perform is to simply re- taken to ensure that the genotype of interest is exposed to the
move the aerial portion of the plant (or an individual leaf) same severity of stress as a wild-type control.
from the roots and measure the decline in fresh weight over An example of one of the complexities of soil drying
time (Figure 4a). The experiment should be set up under experiments is the evaluation of mutants or transgenic
controlled temperature, light and humidity conditions that plants with decreased stomatal conductance or decreased
allow a gradual decline in leaf water content to be observed. growth and leaf area. When water is withheld and the
A decline to 50% water content over the course of 6 to 8 h is condition of the plants assessed after a given time, plants
typical in Arabidopsis (Figure 4a). The rate of water loss is that have reduced stomatal conductance or reduced leaf
largely determined by stomatal conductance; thus, experi- area can be expected to deplete soil water more slowly
ments on leaf water loss measure avoidance of low ww and (avoidance of low ww) and may exhibit delayed wilting
are typically not applicable to investigation of tolerance compared with wild-type plants. Such delayed wilting has
mechanisms. In addition to leaf water loss experiments, been used to label such plants as stress or drought tolerant
measurements of leaf conductance and direct microscopic when instead the transgenic plant has avoided low-ww stress
observation of stomatal apertures in leaf epidermal strips by using the available water more slowly. In general, to
(see for example Leymarie et al., 1999) can be performed. establish whether a particular genetic modification leads to
Rates of leaf water loss can also be estimated based on leaf tolerance of low ww, it must be shown that the stress
temperature. Thermal imaging has been used to isolate response under study differs in plants exposed to the same
Arabidopsis mutants with altered stomatal regulation and severity of stress (same ww) and that this difference leads to
stress avoidance (Merlot et al., 2002; Wang et al., 2004) and a desirable change in phenotype. A better-defined use of the
at the field level to estimate plant water status (Cohen et al., term ‘tolerance’, as well as other terms related to the low-ww
2005). response, could do much to clarify the literature on this
Because stomatal conductance is controlled in large part topic.
by ABA, measurements of leaf water loss are often most These difficulties can be overcome in two ways. The first
useful as an indicator of altered accumulation of or sensi- is by quantification of leaf and/or soil ww during the drying
tivity to ABA. Mutants deficient in ABA and many (although cycle. This can be combined with control of humidity levels
not all) mutants with altered ABA sensitivity exhibit altered or partial rewatering of some plants to ensure that the
leaf water loss. In our laboratory, leaf water loss experiments comparisons of stress response are made only between
are followed by, or performed concurrently with, other tests plants exposed to the same ww (see for example: Sharp
of ABA accumulation and response. These include the effect et al., 2000; Thompson et al., 2004). Partial rewatering can
of ABA on seed germination and seedling growth and ABA- also be used to extend the time for which the plants are
dependent gene expression and stress-induced accumula- exposed to low ww, thus allowing physiological and molecu-
tion of ABA. In many cases, these parameters are measured lar responses to low ww be examined in more detail. These
using the polyethylene glycol (PEG)-infused agar plate experiments are particularly relevant to more detailed
system described below. Measurements of ABA-responsive evaluation of crop species (Sharp et al., 2000; Thompson
seed germination have been described in numerous studies et al., 2004) and numerous other studies where parameters
(see for example Finkelstein, 1994) and typically involve such as osmotic adjustment and leaf growth have been
plating seed on media containing ABA at a range of evaluated in a number of crop species (see for example Babu
concentrations and scoring either emergence of radicles or et al., 1999; Puliga et al., 1996).
the formation of green cotyledons over a period from 1 to In the case of Arabidopsis, however, repeated measure-
10 days after the end of stratification. ments of leaf or soil ww during the drying cycle are
laborious and require a quantity of material that may be
difficult to obtain. For genetic studies, where a mutant or
Soil drying
transgenic plant is being compared with a wild type, the
Soil drying experiments using pot-grown plants are typically easiest way to ensure a valid comparison while avoiding
done by removing the water supply and measuring some extensive measurements of ww is to grow the wild-type
aspect(s) of plant growth, survival and water status after a plant in the same pot as the genotype under evaluation
fixed period of soil drying. Such soil drying experiments can (Figure 4b). Thus the roots of both genotypes will grow
at first seem quite straightforward but often turn out to be one into the same soil and be exposed to the same ww even if
of the most difficult types of experiment to interpret. This is one genotype uses water more quickly than the other. This
because the severity of stress experienced by the plant is not approach can be combined with measurement of soil ww at
determined directly by the investigator but rather by the plant the end of the drying cycle to quantify the final severity of
itself based on the rate at which it depletes the available soil the stress.
ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539
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Methods and concepts in drought, salt and freezing stress 531

The rate of soil drying is a key factor in these experiments. high-molecular-weight solute such as PEG of molecular
A very rapid rate of soil drying allows little time for slow weight 6000 or above. Polyethylene glycol of this molecular
responses such as solute accumulation or cell wall modifi- weight range cannot enter the pores of plant cells (Carpita
cation to occur and can cause many important aspects of the et al., 1979; Oertli, 1985) and thus causes cytorrhysis rather
low-ww response to be overlooked. Using a sufficiently large than plasmolysis. Polyethylene glycol is also a better choice
and deep pot will avoid this situation. The soil type [we for imposing low ww than the often used solute mannitol
typically use a well-aerated potting mix such as Metro-mix because mannitol has been shown to be taken up by plant
350 (Sungrow Horticulture, Bellevue, WA, USA): similar cells and can cause specific toxic effects on growth (Hohl
potting mixtures are also available from other suppliers], and Schopfer, 1991; Verslues et al., 1998). An example of the
humidity, temperature and light intensity will also affect the toxic effects of mannitol and a similar solute melibiose are
rate of drying and these factors must be adjusted empirically shown in Figure 5. For maize primary roots, transfer to a
for any given set of conditions. As a rule of thumb, leaf water )1.6 MPa solution of mannitol or melibiose had less initial
content should decline by no more than 30–40% over a 10– effect (0–10 h) on root growth than transfer to a )1.6 MPa
12-day period after the cessation of watering. PEG solution (Verslues et al., 1998). This is consistent with
Several measurements of response to low ww can be used mannitol and melibiose being taken up by the roots, thus
in conjunction with soil drying experiments. A general leading to less initial loss of turgor and less initial growth
indication of plant performance can be obtained through inhibition. After 48 h, however, PEG-treated roots had
measurements of growth (shoot fresh and dry weights, leaf recovered and resumed steady-state growth, albeit at a
area and root mass after soil removal), efficiency of water reduced rate [root growth of the unstressed control at this
use or photosynthetic performance. Measurement of leaf time was approximately 4 mm h)1 (Verslues et al., 1998)]
relative water content and solute content and calculation of while growth of the mannitol or melibiose roots had
osmotic adjustment have been performed for many crop stopped. This clearly demonstrates that mannitol, and other
species (Babu et al., 1999; Zhang et al., 1999) and allow the low-molecular-weight solutes, have toxic effects that can
capacity for dehydration avoidance to be accessed. If obscure the low-ww response. In experimental systems such
dehydration tolerance is the main interest, then measure- as PEG-infused agar plates (Protocol S1 in the Supplement-
ments of plant survival after severe stress and measure- ary Material accompanying this article) where there is low
ments that quantify cellular damage such as loss of transpiration, root damage is avoided, and the roots are not
chlorophyll content, electrolyte leakage and ROS-induced
damage (see below) can be performed.

Low-ww treatment using PEG-infused agar plates

Many studies of low-ww stress have used osmotica to lower
the ww of plant growth media. This approach has many
advantages: ww can be controlled precisely and reproducibly
and a large number of treatments can be performed quickly.
Osmoticum treatment does, however, bring up its own set of
potential problems that become apparent when osmoticum
treatment is compared with soil drying. In most cases, when
soil water content decreases water is withdrawn from both
the cell wall and the protoplast resulting in cytorrhysis, a
process where both the cell wall and protoplast shrink (Oertli,
1985). This contrasts with the response to low molecular
weight solutes such as mannitol that are often used to lower
ww. In this case the solute freely penetrates the pores of the
cell wall and causes plasmolysis; a loss of water from and
decrease in volume of the protoplast while the volume of the Figure 5. Rates of primary root elongation in maize seedlings transferred
cell wall remains unchanged. Because it is not a part of the from wet vermiculite to )1.7 MPa solutions of either PEG, mannitol or
typical soil drying response and may cause cellular damage melibiose.
In all cases, solutions were oxygenated to prevent root hypoxia (see Verslues
that is perceived and responded to differently from water loss et al., 1998 for methods). Rates of root elongation in seedlings transferred to
caused by soil drying, plasmolysis should be avoided in high-ww (no added solute) solution increased to approximately 4 mm h)1 by
studies of low ww or salinity (Munns, 2002). 50 h (data not shown). Thus, PEG treatment caused a reduction of approxi-
mately 60% in the steady-state root elongation rate but mannitol or melibiose
Experimentally, a cytorrhytic rather than plasmolytic low- of the same ww completely stopped root elongation by 50 h. Data are from
ww treatment can be imposed using solutions containing a Verslues et al. (1998) and Verslues (1997).

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

subjected to hypoxic conditions by submergence in PEG original agar plate and transferred by moving the mesh and
solution. Polyethylene glycol is the best solute that we are seedlings to the PEG-infused plate (Verslues and Bray, 2004;
aware of for imposing a low-ww stress that is reflective of the van der Weele et al., 2000). For ww of )0.7 MPa or below, this
type of stress imposed by a drying soil (Verslues and Bray, transfer leads to rapid dehydration of the seedlings (Versl-
2004; Verslues et al., 1998; van der Weele et al., 2000). ues and Bray, 2004). This loss of water in turn causes a
In addition to the choice of solute used to impose the low number of rapid stress responses including high levels of
ww stress, our experience, and that of others (van der Weele ABA accumulation (Figure 4c) and, similar, to other systems,
et al., 2000), shows that for many types of measurements, it rapid induction of a number of stress- and ABA-regulated
is better to use media without sugar, or with a low level of genes (P. E. Verslues and J.-K. Zhu, unpublished). These
sugar (0.5% or less). This is because sugar is well known to events, which we refer to as the ‘acute’ phase of the low-
affect ABA responses (Finkelstein et al., 2002). Also, the ww response (Figure 4c) have been the focus of most
addition of high a high level of sucrose itself can induce an studies of low-ww response at the molecular and genetic
osmotic response (the ws of a 3.0% sucrose solution is levels. This acute response is followed by longer-term
approximately )0.2 MPa). Thus, seedlings in ‘control media’ responses, such as solute accumulation and osmotic
containing a high level of sucrose can already be experien- adjustment (Verslues and Bray, 2004) and changes in root
cing a low level of osmotic stress. This causes a high and shoot growth (van der Weele et al., 2000) indicative of
baseline level for many low-ww responses. For example, an adjustment to and recovery from the effects of the
ABA levels of more than 300 ng g)1 fresh weight (FW) have reduced ww. These recovery and longer-term responses are
been reported for Arabidopsis seedlings on MS media with also important aspects of the low-ww response to be
3% sucrose (Ruggiero et al., 2004) whereas we routinely investigated by molecular and genetic studies. The PEG-
observe ABA levels of 1 to 4 ng g)1 FW in a half-strength MS infused plate system is in many ways (imposition of a
medium without sucrose (Verslues and Bray, 2004). This constant low ww with minimal transpiration) similar to the
high baseline and the possibility that sugar from the dry vermiculite system that has been used to study low-ww
medium can accumulate in the plant tissue and reduce the responses of seedlings of maize and other crop species
water loss caused by further decreases in ww means that (Sharp et al., 1988, 2004).
many low-ww responses can be difficult to detect in high-
sugar media.
Salt stress
A system of using PEG-infused agar plates to impose
low ww has been described by van der Weele et al. (2000)
Similarities and differences in salt stress, low ww and other
and a modified version of this procedure is in use in our
abiotic stresses
laboratory. A detailed protocol for the preparation and use
of PEG plates is included as Supplementary Material with High salt causes several types of plant stress including
this article (Protocol S1). This system has the advantage of altered nutrient uptake, especially of ions such as Kþ and
being able to easily make plates of a range of ww without Caþ, accumulation of toxic ions, especially Naþ, osmotic
the complications that arise from using low-molecular- stress and oxidative stress. Since NaCl is the major com-
weight solutes. Another advantage is that as long as steps ponent of most saline soils, our usage of the terms salinity
are taken to prevent drying of the plates use of PEG- and salt stress here refers to stress caused by high levels of
infused plates allows the imposition of a constant ww over NaCl. Salt stress differs from the low ww imposed by soil
time. Because ww is constant and transpiration minimal in drying or a high-molecular-weight solute in that a major
the PEG-infused plate system, avoidance of stress is not an factor causing long-term injury in salt stress is the ionic
issue; the seedlings must equilibrate with the ww of the imbalance and toxicity caused by excess Naþ rather than the
agar over time. Thus, the PEG plate system is ideal for effects of salt on ww (Huh et al., 2002; Munns, 2002). Munns
studies of dehydration avoidance and mechanisms of (2002) refers to several studies reporting that rapid re-
dehydration tolerance. Measurements of growth, water sponses to salt (responses that occur within a few hours of
and solute content, hormone accumulation and stress- application of salt) often resemble responses to low ww im-
regulated gene expression are examples of specific traits posed using non-ionic solutes. However, longer-term re-
that can be quantified. sponses that occur over a time frame of days to weeks are
Seeds can be plated directly onto PEG-infused plates and more salt specific. This is also consistent with our isolation
seed germination and growth measured. However, in many of several salt overly sensitive (sos) mutants that are
cases the more useful experiment is to plate seeds on hypersensitive to salt but not to non-ionic osmotic stress and
unstressed media (typically half-strength MS without sugar) regulate a relatively small number of ion transport processes
and transfer them to PEG-infused plates after 5–7 days of and genes specifically involved in tolerance of salt stress
growth (Figure 4c). To facilitate transfer of seedlings be- (Gong et al., 2001; Shi et al., 2002; Wu et al., 1996; Zhu, 2000;
tween plates, seed can be plated on a mesh overlaid on the Zhu et al., 1998).

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

Control Stress absence of transpiration to carry salt to the shoot can both
alter the phenotype). Microarray analysis of salt- and
dehydration-treated plants has also indicated substantial
PEG NaCl differences between the gene expression profiles elicited by
these stresses (Seki et al., 2002). One consideration for both
–0.75 MPa 100 nM low-ww and salt stress experiments should be to identify
factors that are specific to, or more important to, one type of
stress and those that may be shared and are of similar
importance to salt, low ww and other abiotic stresses.
Here again, the concepts of homeostasis and of tolerance
versus avoidance are useful. Salt injury can be avoided by
maintaining proper ion homeostasis. This can be done by
excluding salt from the cytoplasm, either through reducing
salt uptake by the roots, activating salt export or by
compartmentalizing the salt in the vacuole (Munns, 2002;
Zhu, 2003). Under conditions of transpiration, blocking salt
transport from the roots to the shoot is also critically
important. Several lines of evidence suggest that the SOS
signaling pathway, by regulating Naþ and Kþ transport at
both the plasma membrane and tonoplast, has a major role
in maintaining ion homeostasis and thus avoiding salt injury
Figure 6. Growth of Arabidopsis seedlings on control (half-strength MS (Zhu, 2002, 2003). Also, HKT1 is a major determinant of salt
medium with 0.5% sucrose, ww ¼ )0.30 MPa) and PEG-infused or salt-
containing plates, both having ww ¼ )0.75 MPa. Seeds were plated on each tolerance through its role in Naþ uptake and transport of Naþ
medium, stratified at 4C for 3 days and seedlings grown for 7 days on within the plant (Liu et al., 2000; Rus et al., 2001). It is these
vertically oriented plates before photographs were taken. ion homeostasis mechanisms that are most likely to be
specifically important in the salt stress response and of
lesser importance in responses to other abiotic stresses.
A simple example to illustrate some of the possible Other salt responses are important for tolerating the
differences between low ww and salt stress is shown in deleterious effects of high cytoplasmic levels of salt
Figure 6. Arabidopsis seedlings were germinated and grown accumulation or of dehydration. To the extent that they
on low-ww PEG-infused agar plates or salt-containing agar have been characterized, the mechanisms for tolerating
plates. The low-ww and salt treatments used were of the accumulation of salt in the plant tissue are closely related
same ww ()0.75 MPa) and caused a similar amount of total to the mechanisms of tolerating dehydration caused by
inhibition of seedling growth. As previously observed (van low ww or freezing. These mechanisms can include
der Weele et al., 2000), low ww caused a large inhibition of accumulation of compatible solutes and proteins and
shoot growth with root growth being relatively unaffected or ROS detoxification. It is in these tolerance mechanisms
even slightly increased. The relative maintenance of root that many of the commonalities between salt, low ww and
growth at low ww is a well established response to low ww freezing can be found.
(Hsiao and Xu, 2000) and is the result of regulation of growth
by ABA and other factors (Sharp and LeNoble, 2002).
Experimental techniques for evaluating salt stress response
In contrast, seedlings grown in agar plates with 100 mM
NaCl had a greater inhibition of root growth, most likely Salt stress can be imposed by irrigating soil-grown plants
caused by direct toxicity of Naþ. In this case, shoot growth with saline solutions or by transferring seedlings or plants to
was inhibited equally or slightly less than root growth, most salt-containing media. One important consideration is that
likely because the rate of transpiration in the plates is too low plasmolysis should be avoided whenever possible (Munns,
to cause a build-up of high levels of Naþ in the shoot. As has 2002). For pot-grown plants this can be done by adding salt
been previously suggested (Munns, 2002; Zhu, 2003), factors gradually or in steps of 50 mM or less separated by time for
that affect the uptake and distribution of Naþ within the plant the plant to adjust. Pots should be periodically rewatered
can have a predominant role in the response to salt stress. with the same saline solution to keep the salt concentration
Thus, while in a broad sense salt and low ww both have the in the soil at a constant level. Similar to the soil drying
same effect of inhibiting growth and causing cellular dam- experiments described above, it is advisable to grow the
age, the specific changes involved can be different and can genotype being tested in the same pot as a wild-type control
be influenced by the choice of experimental system (in this to ensure that they are exposed to the same salt concen-
example, the amount of sugar in the medium and the tration. Another concern is that the nutrient content of the

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

media should be sufficient such that addition of salt does not similar age from test plants and an appropriate wild-type
cause nutrient deficiency by decreasing the activity of other control are floated in NaCl solution and the extent of
ions, particularly calcium (Cramer et al., 1986; Reid and bleaching and chlorophyll loss determined. In comparing
Smith, 2000). Salt treatment can also be performed by different genotypes, this technique eliminates any effect
incorporating NaCl into agar plates. Seeds can then be ger- of altered root to shoot ion transport and allows a more
minated directly on the salt-containing media or transferred focused assessment of the ability of the tissue to tolerate
to the salt stress plates. For salt-treated plants or seedlings, a Naþ accumulation.
number of traits can be measured to quantify the salt re-
sponse. These most often include measurements of growth
Tissue ion content and uptake
and survival to assess the overall level of salt resistance.
A complete investigation of the effect of a particular genetic
change on the salt stress response should include a quan-
Root and shoot growth, stomatal conductance and
tification of the accumulation of ions in plant tissue. Bulk
photosynthesis
tissue levels of Naþ and other ions of interest can be quan-
The effects of salt can be quantified through effects on tified by straightforward methods such as atomic absorption
growth (root fresh or dry weight, leaf area and leaf expan- spectroscopy. Potassium is of particular interest, as main-
sion and time of flowering and seed yield) and stomatal taining Kþ/Naþ selectivity is critical for salt tolerance (Zhu,
conductance and photosynthetic gas exchange. The specific 2003). If altered Kþ levels are observed, analysis of Kþ uptake
experiments to be performed depend on the trait of greatest can be performed by quantification of radioactive 86Rbþ
interest and the feasibility of the experiments for the number uptake (Wu et al., 1996). Comparison of accumulation of,
of genotypes to be tested. It must be noted that such and growth responses to, Naþ and other ions such as Liþ, a
experiments cannot determine whether any differences ob- toxic Naþ analog, and Csþ, another toxic ion, can differen-
served are caused by altered ion homeostasis (for example tiate between a specific effect on Naþ transport and more
altered shoot Naþ accumulation or Kþ/Naþ ratio) or altered general effects on ion uptake (Zhu et al., 1998; Protocol S2,
tolerance to Naþ accumulation. To answer this question it is Supplementary Material). In general, such measurements
necessary to also quantify tissue ion content and/or ion can address the question of whether a genetic change alters
uptake (see below). the ability of the plant to avoid salt-induced damage by
The most extensive experience of our laboratory is in keeping tissue Naþ levels low while maintaining uptake of
rapidly screening Arabidopsis lines for altered root growth other critical ions. Such analysis of the sos1, -2 and -3 mu-
under salt stress using a root bending assay (see Protocol S2 tants had implicated these loci in the control of Kþ/Naþ ion
in the Supplementary Material). This method was employed homeostasis well before the identities of the mutated genes
to identify sos mutants of Arabidopsis (Liu and Zhu, 1998; were known (Zhu et al., 1998).
Wu et al., 1996). In this method, seeds are plated on control
media (typically MS or half-strength MS) and grown for
Germination
approximately 4 days on vertically oriented plates. Seed-
lings are then transferred to plates containing NaCl (50– Seed germination assays can provide a quick assay of salt
200 mM) and the plates inverted so that the roots point response but must be interpreted with caution. A high rate of
upward. In seedlings that continue to grow after transfer to germination under salt stress is not well correlated with
salt-containing media, the roots will acquire a curled salinity tolerance at later developmental stages (Almansouri
appearance as they grow downward. The advantage of this et al., 2001; Kurth et al., 1986; Saleki et al., 1993). In agar
method is that it allows the extent of root growth to be media with high sucrose, seed germination and initial
checked rapidly without having to mark the position of the growth can occur in the presence of relatively high levels of
root apex. For salt stress, root bending assays have typically salt but is normally blocked by accumulation of ABA. This is
been done in media with high levels of sucrose (up to 3%), as supported by the observation that several mutants that block
high sucrose stimulates root growth and makes it easier to ABA synthesis have increased germination under saline
find mutants with inhibited root growth. Although agravi- conditions (Gonzalez-Guzman et al., 2002; Ruggiero et al.,
tropic mutants will also not exhibit root bending, they can be 2004). We have also observed similar increased germination
easily recognized by continued upward root growth. and growth when the ABA-deficient mutant aba2-1 is ger-
minated on salt- or PEG-infused plates containing 3% su-
crose (P.E. Verslues and J.-K. Zhu, unpublished). Under
Salt-induced leaf damage
most conditions, this inhibition of germination and early
An example of a quick method to measure salt-induced seedling growth by ABA is an adaptive response; it allows
damage is by leaf disk assay (Sanan-Mishra et al., 2005; the plant to delay the start of growth and, importantly,
Singla-Pareek et al., 2003). Leaf disks from leaves of a transpirational water loss, until conditions are more

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

favorable. Screens that have looked for mutants with en- specific cellular structures that can nucleate ice formation
hanced germination and early seedling growth under salt have not been identified (Ashworth and Kieft, 1995).
stress in non-transpiring conditions have predominantly Consequently, sterile leaf disks can be supercooled
found ABA-deficient or ABA-insensitive mutants (Gonzalez- (cooled below freezing without ice nucleation) to )8C
Guzman et al., 2002; Quesada et al., 2000, 2002; Ruggiero whereas leaves colonized by bacteria will nucleate ice
et al., 2004; Saleki et al., 1993; Werner and Finkelstein, 1995). formation at approximately )2C (Lindow et al., 1982).
Whether or not such ABA-deficient mutants should be des- Experimentally, a constant ice nucleation temperature can
cribed as salt tolerant should be carefully considered. In be imposed by incubating plants with ice chips.
addition, it is important to determine the ABA content and After initiation of ice formation, subsequent nucleation
ABA sensitivity of any genotypes that exhibit altered ger- occurs on the surface of the ice crystal itself. In addition to its
mination under saline (or low-ww) conditions before effects on dehydration avoidance (Figure 2) the composition
attempting to interpret their role in stress tolerance. and structure of the cell wall provides the plant with an
opportunity to control the location of ice nucleation sites in
the tissue. At the whole-plant level, ice first forms in the large
Freezing
vessels of the xylem in leaves and stems, in substomatal
The general term ‘cold stress’ can be divided into two related cavities and in intercellular spaces (Levitt, 1980). The large
phenomena; chilling stress and freezing stress. Chilling diameter of xylem vessels favors ice formation, and their
stress occurs at temperatures lower than the plant’s normal dilute sap has a higher freezing point than other solutions in
growth temperatures but not low enough to cause ice for- the plant. Once ice forms it will spread throughout the
mation (Levitt, 1972). Chilling is damaging primarily because vessels and into the extracellular spaces of other tissues.
of membrane leakiness caused by an inability to increase However, the ice crystals cannot penetrate an intact plasma
membrane fluidity to accommodate the lower temperature. membrane to inoculate the cytoplasm. Thus ice formation in
Such chilling-sensitive plants are also highly sensitive to the extracellular space decreases the ww of the extracellular
freezing stress (Guy, 2003). Here we will focus on freezing space, leading to movement of water out of the cells and cell
stress as it is intrinsically related to dehydration caused by walls until equilibrium of ww across the membrane is re-
low ww. In the case of freezing, it is the formation of ice established. This is similar to the cytorrhytic dehydration
crystals in the extracellular space that dehydrates the cell. described above for plants in drying soil, although the extent
Thus, the dehydration tolerance mechanisms discussed of dehydration is likely to be more severe during freezing
above are also relevant to tolerance of freezing stress. For stress. Thus, freezing stress causes damage primarily by
example, the constitutively freezing tolerant mutant eskimo1 dehydrating and collapsing cells, disrupting tissue structure
(esk1; Xin and Browse, 1998) has increased total solute by the formation of large ice crystals and causing large
accumulation and increased accumulation of the compatible fluxes of water across cellular membranes during freezing
solute proline, traits that are also likely to make esk1 more and thawing.
resistant to low-ww-induced dehydration (to our knowledge,
however, this has not been tested). Increased tolerance to
Experimental procedures for imposing freezing stress
dehydration, salt and freezing has been reported in plants
overexpressing DREB (dehydration response element bind- Since the formation of ice is so important for freezing tests, fac-
ing) transcription factors which leads to enhanced expres- tors affecting ice formation should be considered carefully when
laboratory freezing tests are performed. These issues have been
sion of a wide range of stress responsive genes (Liu et al., discussed in detail by Gusta et al. (2003) and are reviewed briefly
1998; Kasuga et al., 1999). In addition to the tolerance of here.
freezing itself, the ability to increase chilling and freezing
tolerance by first exposing the plant to a short duration of a
less severe low-temperature treatment is an area of active Ice nucleation
investigation. Under controlled conditions of plant growth naturally occurring ice
To understand the methodology used to impose freez- nucleators are generally absent and therefore it is important to
ing stress, it is important to understand the mechanism of incubate the plant tissues with ice chips (which act as nucleating
ice formation and its harmful effects on the cellular agents).
environment. Freezing injury is caused by the formation
of ice in and around cells. The temperature at which ice Nucleation temperature
begins to form depends on the presence of ice nucleators.
The temperature at which nucleation is started is important because
In most situations, epiphytic bacteria found on plant
prolonged supercooling results in non-freezing equilibrium (Olien,
leaves provide sites for ice nucleation (Lindow et al., 1974), resulting in explosive ice growth and formation at unfavo-
1982). Plant cells and cell walls may also have intrinsic ice rable sites. This indicates that temperature until which supercooling
nucleation sites but these are not as efficient and the should be done is very important for determining the LT50 (the

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

temperature at which 50% lethality occurs). An example of this was accumulation, the increasing recognition that ROS accumulation
observed in Solanum acaule where the LT50 of leaves was deter- is an important aspect of abiotic stress signaling must be kept in
mined to be )7C, if nucleation was started at )1C. However, when mind. Total ROS accumulation can be assayed using the non-
leaves were supercooled to )2C, followed by ice nucleation the fluorescent dye 2¢,7¢-dichlorodihydrofluorescein diacetate
LT50 was observed to be )3C (Rajashekar et al., 1983). Before (H2DCFDA) that is oxidized to the highly fluorescent 2¢,7¢-dichlo-
beginning freezing experiments, it is advisable to review the freez- rofluorescein (DCF). The non-fluorescent H2DCFDA can diffuse
ing stress literature for a particular species to determine if an opti- readily into cells but becomes trapped after interaction with ROS
mal ice nucleation temperature has been established. molecules and oxidation to DCF. The DCF can then be detected
by confocal scanning fluorescence microscopy (Mazel et al.,
2004). Superoxide can be specifically detected by nitroblue
Intactness of the cuticle tetrazolium (NBT) staining (Lee et al., 2002). Hydrogen peroxide
can be detected either by staining using 3¢,3¢-diaminobenzidine
The cuticle acts as a barrier to ice formation inside the plant tissue (Lee et al., 2002) or by quantitative assay of tissue extracts using
(Wisniewski and Fuller, 1999). Damage to the cuticle, such as the hydrogen peroxide-specific dye Amplex Red (Shin and
mechanical damage or damage caused by pathogen infection, can
Schachtman, 2004). The most common measure of ROS-induced
result in lesions through which ice crystals can grow and can skew
damage is lipid peroxidation. Lipid peroxidation can be estimated
the results of freezing tests. Also, when excised tissues are used for by the formation of thiobarbituric acid reactive substances
freezing stress, the cut surface provides an excellent opportunity for (TBARS) and quantified in terms of malonaldehyde (Heath and
ice to enter the conducting vessels. Because of this, such an assay
Packer, 1968). It has been observed that some salt-tolerant
may not accurately reflect the whole-plant response where such an
germplasms have less peroxidative damage than more sensitive
easy route for ice entry is not available. genotypes (Shalata and Tal, 1998).

Cooling rate Conclusions

The rate of cooling is another important criterion to be considered in There is an increasing availability and ease of generation of
artificial freezing tests. A rapid rate of cooling can result in non-
genetically modified lines in Arabidopsis, and other model
uniform cooling across the plant tissue and rapid freezing that does
organisms, that either increase (overexpression or ectopic
not mimic the natural freezing process. If the rate of cooling is too
expression) or decrease (mutants, gene knockouts and RNA-
slow, it may be more difficult to detect differences in freezing tol- interference lines) the production of certain gene products. Thus,
erance.
the genetic resources available for the investigation of abiotic
Many freezing protocols have been developed for a number of
stress resistance have increased dramatically in the last few
plant species. The Supplementary Material to this paper includes a years and are likely to continue to do so. This has led to the
protocol (Protocol S3) suitable for Arabidopsis. It can be adapted for emergence of what has been termed the ‘phenotype gap’ (Miflin,
other species and conditions with consideration of the factors
2000), where the identification of useful phenotypes and appli-
outlined above.
cations has increased at a much slower pace than the increase in
molecular and genetic data. It is hoped that this paper will sti-
mulate thinking about the best methods to use to translate the
Quantifying abiotic stress-induced cellular damage
increasingly available molecular and genetic resources into
Severe levels of low-ww, salt or freezing stresses cause cellular identification and better understanding of the phenotypes asso-
damage, and quantifying the extent of this damage can be an ciated with abiotic stress resistance.
important component in testing the effect of a specific genetic
modification. Often, the extent of stress-induced damage is
measured by testing the percentage of plants that survive and Acknowledgements
recover after undergoing a stress treatment and then being Research in our laboratory has been supported by grants from US
transferred back to unstressed conditions. Such survival tests can
National Science Foundation, Department of Agriculture and
be done quickly, and in many cases are sufficient to detect dif- National Institutes of Health. Funding for this paper was provided in
ferences between genotypes. However, such tests are a relatively part by US National Science Foundation grant IBN-0420152. P.E.V. is
crude measure of the stress response and can miss differences in supported by an NIH postdoctoral fellowship (1F32GM074445-01).
cellular damage that are significant but do not change the ability
We thank Rebecca Stevenson for assistance in preparing the
of the whole plant to recover after release of the stress. In many manuscript.
cases the particular gene, protein or cellular component under
study will suggest specific methods for quantifying the damaging
effects of low ww. In other cases, assays of electrolyte leakage, Supplementary Material
ROS accumulation and ROS-induced chemical damage can be
good indicators of the degree of cellular damage. Electrolyte The following supplementary material is available for this article
leakage allows relatively quick assessment of the intactness of online:
cell membranes. Detailed procedures for measuring electrolyte Appendix S1. Protocol I: Preparation of PEG-infused plates for low
leakage in freezing and low-ww treated tissue are included with water potential treatment.
the Supplementary Material accompanying this article (Protocol Appendix S2. Protocol II: Evaluation of salt tolerance in Arabidop-
S4). sis seedlings by measuring root elongation.
Accumulation of ROS and ROS-induced damage can serve as Appendix S3. Protocol III: Monitoring plant survival by whole plant
an indicator both of structural damage to cells and of metabolic freezing tests.
dysfunction. However, when interpreting the effects of ROS Appendix S4. Protocol IV: Electrolyte leakage test.

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

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The Plant Journal - 2006 - Verslues - Methods and Concepts in Quantifying Resistance To Drought Salt and Freezing Abiotic

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The Plant Journal - 2006 - Verslues - Methods and Concepts in Quantifying Resistance To Drought Salt and Freezing Abiotic

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The Plant Journal (2006) 45, 523–539 doi: 10.1111/j.1365-313X.2005.02593.

TECHNIQUES FOR MOLECULAR ANALYSIS

Methods and concepts in quantifying resistance to drought,

Received 23 May 2005; revised 4 August 2005; accepted 6 September 2005.

Keywords: Arabidopsis, stress quantification.

ª 2006 Blackwell Publishing Ltd 523

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

Figure 1. Conceptual diagram of the stress

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

Figure 2. (a) Possible values of ww, ws (solute

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

Experimental techniques for evaluating the low-ww

Here we describe some basic experimental designs

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

Figure 4. Three types of experiments used to evaluate low-ww responses.

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

water. This can lead to confusion if the severity of the stress is

Low-ww treatment using PEG-infused agar plates

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

Cooling rate Conclusions

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

ª Blackwell Publishing Ltd, The Plant Journal, (2006), 45, 523–539

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