Recent advances in engineering plant tolerance to abiotic stress:
achievements and limitations
Basia Vinocur and Arie Altman
Abiotic stresses, especially salinity and drought, are the
primary causes of crop loss worldwide. Plant adaptation to
environmental stresses is dependent upon the activation of
cascades of molecular networks involved in stress perception,
signal transduction, and the expression of specific stressrelated genes and metabolites. Consequently, engineering
genes that protect and maintain the function and structure
of cellular components can enhance tolerance to stress. Our
limited knowledge of stress-associated metabolism remains a
major gap in our understanding; therefore, comprehensive
profiling of stress-associated metabolites is most relevant to
the successful molecular breeding of stress-tolerant crop
plants. Unraveling additional stress-associated gene
resources, from both crop plants and highly salt- and droughttolerant model plants, will enable future molecular dissection of
salt-tolerance mechanisms in important crop plants.
Addresses
The Robert H Smith Institute of Plant Sciences and Genetics in
Agriculture and the Otto Warburg Center for Agricultural Biotechnology,
The Hebrew University of Jerusalem, PO Box 12, Rehovot 76100, Israel
Corresponding author: Altman, Arie ([email protected])
Current Opinion in Biotechnology 2005, 16:123–132
This review comes from a themed issue on
Plant biotechnology
Edited by Dirk Inzé
Available online 11th February 2005
0958-1669/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2005.02.001
responsive mechanisms to re-establish homeostasis and to
protect and repair damaged proteins and membranes
(Figure 1) [2]. In contrast to plant resistance to biotic
stresses, which is mostly dependent on monogenic traits,
the genetically complex responses to abiotic stresses are
multigenic, and thus more difficult to control and engineer. Plant engineering strategies for abiotic stress tolerance [2] rely on the expression of genes that are involved
in signaling and regulatory pathways [3,4] or genes that
encode proteins conferring stress tolerance [5] or
enzymes present in pathways leading to the synthesis
of functional and structural metabolites [6–8]. Current
efforts to improve plant stress tolerance by genetic transformation have resulted in several important achievements; however, the genetically complex mechanisms
of abiotic stress tolerance makes the task extremely
difficult. For this reason, biotechnology should be fully
integrated with classical physiology and breeding [2,9].
Several comprehensive reviews on molecular mechanisms of abiotic stress tolerance and on engineering tolerance to stress, mainly profiling a large number of stressassociated genes and signal-transduction systems, have
been recently published [3,10]. Several successful
approaches to achieving tolerance through the genetic
engineering of specific genes have been reviewed [2] and
some pitfalls were also discussed [9]. In the present
review, selected recent advances in our understanding of
abiotic stress tolerance are presented, with special
emphasis on several downstream processes. The use of
model plants and the interacting factors in molecular
breeding for crop tolerance to abiotic stress will also be
discussed.
Stress-associated genes and proteins:
expression and proteomics
Introduction
In the face of a global scarcity of water resources and the
increased salinization of soil and water, abiotic stress is
already a major limiting factor in plant growth and will
soon become even more severe as desertification covers
more and more of the world’s terrestrial area. Drought and
salinity are already widespread in many regions, and are
expected to cause serious salinization of more than 50% of
all arable lands by the year 2050 [1]. In a world where
population growth exceeds food supply, agricultural and
plant biotechnologies aimed at overcoming severe
environmental stresses need to be fully implemented.
Plant adaptation to environmental stresses is controlled
by cascades of molecular networks. These activate stresswww.sciencedirect.com
Stress-associated genes and proteins for each of the downstream steps can be grouped into major categories [2].
Some of these are discussed below (Figure 2). The role of
ion and water transporters is not discussed here, as it was
presented by us in an earlier review [2].
Signaling cascades and transcriptional control
Genes involved in signaling cascades and in transcriptional control, such as mitogen-activated protein (MAP)
[11] and salt overly sensitive (SOS) [12] kinases, phospholipases [13] and transcription factors (e.g. heat shock
factor [HSF] and the C-repeat-binding factor /dehydration-responsive element binding protein [CBF/DREB]
and ABA-responsive element binding factor/ABA-responsive element [ABF/ABRE] families) [10], have been
Current Opinion in Biotechnology 2005, 16:123–132
124 Plant biotechnology
Figure 1
Drought
Salinity
Chemical
pollution
Heat
Cold
Secondary stress
Osmotic stress
Oxidative stress
Disruption of osmotic and
ionic homeostasis;
damage of functional and
structural proteins and
membranes
Signal sensing,
perception and
transduction
Osmosensors (e.g. AtHK1),
phospholipid-cleaving enzymes (e.g. PLD), second
messengers (e.g. Ca2+, PtdOH, ROS), MAP kinases,
Ca2+ sensors (e.g. SOS3), calcium-dependent protein
kinases (e.g. CDPKs)
Transcription factors
(e.g. CBF/DREB, ABF, HSF, bZIP, MYC/MYB)
Transcription control
Chaperone functions
(Hsp, SP1, LEA, COR)
Detoxification
(SOD, PX)
Stress-responsive
mechanisms
Gene activation
Osmoprotection
(proline, GlyBet,
sugar polyols)
Water and ion
movement (aquaporin,
ion transporter)
Re-establishment of cellular homeostasis,
functional and structural protection of
proteins and membranes
Stress tolerance or resistance
Current Opinion in Biotechnology
The complexity of the plant response to abiotic stress [2]. Primary stresses, such as drought, salinity, cold, heat and chemical pollution, are
often interconnected and cause cellular damage and secondary stresses, such as osmotic and oxidative stress. The initial stress signals
(e.g. osmotic and ionic effects or changes in temperature or membrane fluidity) trigger the downstream signaling process and transcription
controls, which activate stress-responsive mechanisms to re-establish homeostasis and to protect and repair damaged proteins and membranes.
Inadequate responses at one or more steps in the signaling and gene activation process might ultimately result in irreversible changes in cellular
homeostasis and in the destruction of functional and structural proteins and membranes, leading to cell death. Abbreviations: ABF, ABRE binding
factor; AtHK1, Arabidopsis thaliana histidine kinase-1; bZIP, basic leucine zipper transcription factor; CBF/DREB, C-repeat-binding factor/
dehydration-responsive binding protein; CDPK, calcium-dependent protein kinase; COR, cold-responsive protein; Hsp, heat shock protein;
LEA, late embryogenesis abundant; MAP, mitogen-activated protein; PLD, phospholipase D; PtdOH, phosphatidic acid; PX, peroxidase; ROS,
reactive oxygen species; SOD, superoxide dismutase; SP1, stable protein 1.
extensively studied. However, most of these studies
involved short-term experiments, which cannot provide
conclusions as to the actual stress tolerance of crops. This
is dependent on longer term plant performance with
respect to biomass, yield data and the degree of recovery
from stress. The following discussion focuses only on
those studies where signal transduction and transcription
factors were assessed over a longer term. It has been
Current Opinion in Biotechnology 2005, 16:123–132
recently shown that the constitutive expression of the
tobacco mitogen-activated protein kinase kinase kinase/
Nicotinia protein kinase 1 (MAPKKK/NPK1) in maize
activates an oxidative signal cascade and leads to cold,
heat, and salinity tolerance in the transgenic plants [11].
The transgenic maize maintained significantly higher
photosynthetic rates, suggesting that NPK1 induces a
mechanism that protects the photosynthetic machinery.
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Engineering plant tolerance to abiotic stress Vinocur and Altman 125
Figure 2
Stress-associated genes and proteins
Plant
breeding and
molecular
markers
(e.g. QTLs)
Signaling
Hsps/
pathway
chaperones
components,
and LEA
transcription
proteins
factors
Ion
and water
transport*
ROS
scavenging
and
detoxification
Genetic
transformation
Acquired plant stress tolerance
Other stressOsmolytes,
response
Carbon
Polyamines
osmometabolism mechanisms*
protectants
(e.g.
apoptosis)
Stress-associated metabolites
Current Opinion in Biotechnology
Acquired plant stress tolerance can be enhanced by manipulating stress-associated genes and proteins and by overexpression of stressassociated metabolites. Plant resistance to abiotic stress is a multigenic trait, depending on the combination of many genes, proteins and
metabolic pathways all playing in concert. Stress-associated mechanisms that are not discussed in the present review are marked by an
asterisk. Acquired plant tolerance to abiotic stress can be achieved both by genetic engineering and by conventional plant breeding combined
with the use of molecular markers and quantitative trait loci (QTLs). Hsp, heat shock protein; LEA, late embryogenesis abundant; ROS, reactive
oxygen species.
Most current genomic research is carried out at the DNA
and RNA levels, assuming that gene up- or downregulation will explain abiotic stress processes. However, there
has been an increasing number of demonstrations of
translational and post-translational regulation of stressassociated proteins in response to environmental stress.
For example, using comparative proteomic analysis of the
Arabidopsis root microsomal fraction, a novel component
of salt stress signaling, annexin 1 (AnnAt1), was found to
be upregulated upon stress [14]. AnnAt1 is a member of
a multigene family of Ca2+-dependent membrane-binding osmotic stress-responsive proteins. Interestingly, the
AnnAt1 gene transcript was constitutively expressed,
while protein expression was upregulated in response
to stress and the protein translocated from the cytosol
to the membrane. The mutant plants annAt1 and annAt4
were found to be hypersensitive to salt and abscisic acid
(ABA) during seed germination and early seedling
growth.
Heat-shock proteins and chaperones
Heat-shock proteins (Hsps) and molecular chaperones, as
well as late embryogenesis abundant (LEA) protein
families, are involved in plant abiotic stress tolerance
[2,5]. High temperature, salinity and drought stress
can cause denaturation and dysfunction of many proteins.
We thus expect that Hsps and LEA proteins help to
protect against stress by controlling the proper folding and
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conformation of both structural (i.e. cell membrane) and
functional (i.e. enzymes) proteins (see also Update).
Indeed, positive correlations between the levels of
several Hsps and stress tolerance have been described
[5,15,16]. The increase in Hsp expression under conditions of abiotic stress was studied extensively by functional genomics and proteomics in different plant species
[16–18]. A significant osmoprotective effect was obtained
in Escherichia coli transformed with the cytosolic chaperonin CCP-1a from Bruguiera sexangula, [19]. Studies on
plant genetic transformation with Hsp genes have dealt
mostly with heat stress and thermotolerance. Overexpression of HSP101 from Arabidopsis in rice plants resulted in
a significant improvement of growth performance during
recovery from heat stress [20]. Overexpression of LEA
proteins was correlated in several cases with desiccation
tolerance, although the actual function of these proteins is
still unknown [21]. Recently, overexpression of HVA1, a
group 3 LEA protein isolated from barley (Hordeum
vulgare L.), confering dehydration tolerance to transgenic
rice plants was reported [22].
Reactive oxygen species
Stress-induced production of reactive oxygen species
(ROS) is another aspect of environmental stress in plants
[23]. Alleviation of oxidative damage by the use of
different antioxidants and ROS scavengers can enhance
plant resistance to salt and drought. Transgenic tobacco
Current Opinion in Biotechnology 2005, 16:123–132
126 Plant biotechnology
plants overexpressing Chlamydomonas glutathione peroxidase in the cytosol and in the chloroplast displayed
increased tolerance to oxidative stress, which was
imposed using methylviologen, chilling and salt stress
[24]. Overexpression of the aldehyde dehydrogenase
AtALDH3 gene in Arabidopsis conferred tolerance to
drought and salt stress [25]. The transgenic plants showed
improved tolerance to dehydration, as well as to other
types of stress (salt, heavy metals and hydrogen peroxide),
suggesting that aldehyde dehydrogenase can maintain
membrane integrity under osmotic stress. Aldehyde
dehydrogenase catalyzes the oxidation of toxic aldehydes,
which accumulate as a result of side reactions of ROS with
lipids and proteins [26].
Stress-associated changes in metabolites
and metabolomics
Severe osmotic stress causes detrimental changes in cellular components. A wide range of metabolites that can
prevent these detrimental changes have been identified,
including amino acids (e.g. proline), quaternary and other
amines (e.g. glycine-betaine and polyamines) and a
variety of sugars and sugar alcohols (e.g. mannitol and
trehalose) (Figure 2). Two general strategies for the
metabolic engineering of abiotic stress tolerance have
been proposed: increased production of specific desired
compounds or reduction in the levels of unwanted (toxic)
compounds [27]. However, modulation of a single enzymatic step is usually regulated by the tendency of cell
systems to restore homeostasis, thus limiting the potential
of this approach. Targeting multiple steps in the same
pathway could help to control metabolic fluxes in a more
predictable manner [28].
Amino acids
Proline accumulation was correlated with improved plant
performance under salt stress. Proline-level increments
can be achieved in planta by overexpressing D1-pyrroline5-carboxylate synthetase (P5CS), as found, for example,
in tobacco [28]. This approach also resulted in the
upregulation of proline dehydrogenase, however, which
reduces proline levels. Indeed, Arabidopsis transformation
with proline dehydrogenase antisense [29] or a knockout
of this enzyme [30] resulted in increased free proline
accumulation and better growth performance under salt
stress. To examine the possibility that plant growth
reduction in response to osmotic stress might actually
result from osmolyte accumulation, proline levels were
manipulated by expressing mutated derivatives of P5CS
from tomato in Saccharomyces cerevisiae [31]. The levels of
proline accumulation and cell growth were inversely
correlated in cells grown under normal osmotic conditions. Alternatively, proline might confer a protective
effect by inducing stress-protective proteins. Exogenously applied proline and/or salt stress in Pancratium
maritimum were found to induce the expression of
ubiquitin, antioxidative enzymes, and dehydrins [32].
Current Opinion in Biotechnology 2005, 16:123–132
Amines
Glycine-betaine is a widely studied osmoprotectant, the
accumulation of which has been studied with respect to
modifications of several metabolic steps. Betaine aldehyde decarboxylase from the halophyte Suaeda liaotungensis was introduced into tobacco plants and the in vitro
plantlets were significantly resistant to salt conditions
[33]. Similar results were obtained by transforming rice
with the choline dehydrogenase gene (codA) from Arthrobacter globiformis; the gene product of codA catalyzes the
oxidation of choline to glycine betaine via betaine aldehyde as intermediate. The transgenic rice plants recovered from salt stress and set seeds, in contrast to wild-type
plants [34]. Tomato plants transformed with a bacterial
codA gene targeted to the chloroplast were highly tolerant
to chilling and oxidative stress, showing an increase in
photosynthetic rate, plant survival, flower retention and
fruit set [6]. Co-targeting multiple steps in the same
pathway was found to be a successful strategy for
overexpressing glycine-betaines in plants and bacteria.
Indeed, stress tolerance was enhanced by the genetic
engineering of E. coli and tobacco plants with the betaine
aldehyde–choline dehydrogenase fusion protein [35].
Plant polyamines have previously been shown to be
involved in plant response to salinity [36]. More recently,
genetic engineering for increased biosynthesis of several
specific polyamines resulted, in several cases, in stresstolerant plants [27]. Overexpression of arginine decarboxylase (ADC), ornithine decarboxylase and S-adenosylmethionine decarboxylase induced a significant
increment in putrescine levels and a small increase in
spermidine and spermine levels. Transgenic rice plants
expressing Datura stramonium ADC under the control of
the monocot Ubi-1 promoter produced much higher levels
of putrescine under drought stress only, promoting spermidine and spermine synthesis and ultimately protecting
the plants from drought [37]. Overexpression of spermidine synthase cDNA from Cucurbita ficifolia in Arabidopsis
thaliana significantly increased spermidine levels, and
consequently enhanced tolerance to various stresses [38].
Sugars and sugar alcohols
Overall carbon metabolism and the levels of specific
sugars are severely affected by abiotic stress. In Setaria
sphacelata, a naturally adapted C4 grass, photosynthetic
carbohydrate content was studied under conditions of
both rapid and slow water deficit [39]. In short-term stress
experiments, a decrease in sucrose and starch content was
observed. In long-term experiments, a higher amount of
soluble sugars and a lower amount of starch were found
under stress. The shift of metabolism towards sucrose
might occur because starch synthesis and degradation are
more affected than sucrose synthesis [39]. Trehalose, a
rare, non-reducing sugar, is present in many bacteria and
fungi and in some desiccation-tolerant higher plants. Rice
tolerance to multiple abiotic stresses through engineering
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Engineering plant tolerance to abiotic stress Vinocur and Altman 127
trehalose overexpression was reported [40,41]. The modest increase in trehalose levels in the transgenic plants
resulted in a higher photosynthetic rate and in a decrease
in photo-oxidative damage during stress. Trehalose is
thought to protect biomolecules from environmental
stress, as suggested by its reversible water-absorption
capacity to protect biological molecules from desiccation-induced damage. The low levels of trehalose in
transgenic plants can be explained by specific trehalase
activity, which degrades trehalose; hence, it might be
possible to increase trehalose accumulation by downregulating trehalase activity [42]. Mannitol is another sugar
alcohol that accumulates upon salt and water stress and
can thus alleviate abiotic stress. Transgenic wheat expressing the mannitol-1-phosphatase dehydrogenase gene
(mtlD) of E. coli was significantly more tolerant to water
and salt stress [43]. The transgenic wheat plants showed
an increase in biomass, plant height and number of tillers
(secondary stems in grasses). However, the amount of
accumulated mannitol was too small to account for its
effect as an osmolyte, and the authors suggested that it
might act as a ROS scavenger.
Metabolic profiling
Overall metabolic profiling of plants under stress is an
important tool to study stress-induced changes in metabolites. Arabidopsis metabolic profiling revealed that
plants subject to a combination of drought and heat stress
accumulate sucrose and other sugars, such as maltose and
glucose. By contrast, proline, which accumulated in plants
subjected to drought, did not accumulate in plants during
a combination of drought and heat stress. Heat stress was
found to reduce the toxicity of proline, suggesting that
during the more severe, combined stress, sucrose replaces
proline in plants as the major osmoprotectant [44].
Model plants can be employed to study
abiotic stress tolerance
All major crops are sensitive to salinity and sophisticated
approaches for the molecular breeding of salt-tolerant
crops are therefore needed. These will require the unraveling of additional stress-associated gene resources, from
both crop plants and model plant species that are highly
salt-tolerant. Most of the recent molecular studies on
plant stress tolerance have used Arabidopsis (in addition
to a limited number of crop plants), a typical glycophyte
that is not adapted to salt or drought stress. A substantial
number of halophytes (adapted to salt), as well as
xerophytes (adapted to drought) or desiccation-tolerant
plants, can use mechanisms of stress tolerance that are
similar to those found in glycophytes. Some of these
model plants are mentioned below.
Model plants for studying drought tolerance
Regular drought-tolerant plants can withstand moderate
dehydration conditions of about 30% water loss. By contrast, desiccation-tolerant plants (generally referred to as
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resurrection plants) are tolerant to further cell dehydration, around 90% water loss, and also have the ability to
rehydrate successfully. Resurrection plants have been
widely used as model plants for dehydration studies
[45]. The physiological basis of desiccation tolerance in
resurrection plants is complex, and some mechanisms
might vary between species. These studies suggest that
desiccation tolerance in the vegetative tissues of C. plantagineum is unlikely to result from the presence of genes
that are unique to resurrection plants, asthe relevant
genes are also present in the genome of non-tolerant
plants (e.g. LEA proteins [46]). Thus, the difference
between desiccation-tolerant and non-tolerant plants is
likely to reside in the expression patterns of the genes and
is, in part, a quantitative characteristic [47].
Model plants for studying salinity tolerance
The nature of salinity tolerance was studied in several
halophytes. Mesembryanthemum crystallinum (ice plant) is a
model C3/CAM (Crassulacean Acid Metabolism) halophyte that turned out to be a favorable model plant. Saltstress response mechanisms in the ice plant were studied
with respect to both the salt-stress-induced C3/CAM shift
and the consequent oxidative stress [48], and through the
characterization of Na+/K+ transporters [49] and aquaporins [50]. Interestingly, it was demonstrated that the C3/
CAM shift effectively protects M. crystallinum against
oxidative damage caused by simultaneous salinity and
ozone treatments [48].
Thellungiella halophila (salt cress) is an extreme halophyte
(Figure 3), which was recently shown to be a most
amenable model plant for investigating tolerance to salinity stress. T. halophila is closely related to A. thaliana and
expressed sequence tag analysis revealed 90–95% nucleotide identity in transcripts for well-known housekeeping
genes, suggesting the presence of paralogous genes [51].
In contrast to Arabidopsis, however, Thellungiella tolerates
extreme salinity, cold and drought. Furthermore, T. halophila shares many of the advantages of A. thaliana as an
experimental system. It has a small genome (less than
twice the size of the A. thaliana genome), a short life cycle,
abundant seed production, and is amenable to transformation. High sequence homology at the cDNA level
allows the use of Arabidopsis microarrays for expression
profiling of Thellungiella. Initial transcript profiling experiments [51,52] have revealed that a similar level of
salinity induces fewer genes in Thellungiella than in Arabidopsis; however, under salt-free conditions, some Thellungiella orthologs of stress-related Arabidopsis genes
exhibit high base levels of expression. Salt tolerance in
Thellungiella is associated with specific features of ion
transport, including high K+/Na+ selectivity of ion uptake
into root cells and K+/Na+ exchange between leaf epidermal and mesophyll cells. In addition, ion channels
in Thellungiella root cells have higher K+/Na+ specificity
than the respective Arabidopsis channels [51,53]. This
Current Opinion in Biotechnology 2005, 16:123–132
128 Plant biotechnology
Figure 3
Arabidopsis
thaliana
Thellungiella
halophila
0 mM
200 mM
600 mM
Current Opinion in Biotechnology
Thellungiella halophila is an ideal model plant for studying salinity tolerance. T. halophila is a small halophyte that is highly tolerant to salt stress.
It has a short life cycle and shares many biological and molecular similarities with Arabidopsis thaliana. The photograph shows the performance
of T. halophila and A. thaliana plants grown for seven days in potting medium saturated with 0 mM, 200 mM and 600 mM NaCl. (Original
photograph by A Altman.)
observation was supported by X-ray microanalysis of ions
in root sections, where a gradient of K+ and Na+ was
identified from the epidermal tissues to the central cylinder, and also by the ability of T. halophilla to exclude Cl
ions (A Altman, unpublished). Total sugars, as well as
specific levels of glucose, fructose and mannitol and
several amino acids were found to accumulate to very
high levels in shoots of T. halophila during salt exposure
[51] (A Altman, unpublished).
Applied biotechnology: interacting factors in
molecular breeding for crop tolerance
The improvement of crop abiotic stress tolerance by
classical breeding is fraught with difficulties because
of the multigenic nature of this trait. Further complications arise from the large variability in stress sensitivity at
different periods during the life cycle of a given plant. Of
the various general types of plant response to salinity and
drought stress, avoidance mechanisms mainly result
from morphological and physiological changes at the
whole-plant level. These are less amenable to practical
manipulations. By contrast, tolerance mechanisms are
caused by cellular and molecular biochemical modifications that lend themselves to biotechnological manipulation. The interacting factors are schematically
presented in Figure 4. All types of abiotic stress evoke
cascades of physiological and molecular events and some
of these can result in similar responses; for example,
drought, high salinity and freezing can all be manifested
at the cellular level as physiological dehydration. A full
elucidation of abiotic stress tolerance mechanisms, and
an intelligent breeding strategy for stress tolerance,
require clear and fact-based answers to a number of
questions. Which genes and proteins are upregulated
Current Opinion in Biotechnology 2005, 16:123–132
or downregulated by the different types of abiotic stresses? What are the functions of these stress-responsive
genes and proteins? And, which can be used as genetic
markers for the breeding and selection of stress-tolerant
genotypes or otherwise successfully engineered in
transgenic plants?
The application of quantitative trait loci (QTL) mapping
is one approach to dissecting the complex issue of plant
stress tolerance. When fully developed, this approach will
be of great significance to breeding for abiotic stress
tolerance in plants [9]. QTLs associated with abiotic
stress tolerance have been identified in many important
crop species (e.g. salt stress in rice [54], drought stress in
cotton [55], and cold stress in the woody plant Salix [56]).
A high salt-tolerant indica rice variety was crossbred with a
susceptible japonica rice variety [54] and QTLs were
detected for seedling survival under stress, which correlated well with the degree of leaf damage and Na+
accumulation in the shoots. Frost-susceptible and frostresistant clones of Salix were crossed and frost tolerance at
different time points was assessed by the number of new
leaves, shoot-tip abscission, dry and fresh weight, height
increment, ion leakage and visual injury [56]. The associated QTLs were different at different time points,
suggesting an independent genetic relationship between
non-acclimated and acclimated Salix.
From an evolutionary point of view, all plant responses to
stress and all tolerance mechanisms are programmed and
genotype-specific. However, although adaptation to stress
has some ecological advantages, its metabolic energy
costs can result in yield penalties, consequently limiting
their benefit for agricultural plants. Therefore, efficient
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Engineering plant tolerance to abiotic stress Vinocur and Altman 129
Figure 4
Osmotic stress
Other stresses
Desiccation (water deficit)
Water and soil salinity
Cold (cell freezing)
Ion toxicity (metals, Na/Cl)
heat, anaerobiosis (annoxia)
oxidation, UV, wounding
Impaired metabolism
Gene activation
Structural and functional
proteins and metabolites
Tolerance
Cellular and molecular
mechanisms
ROS generation
Growth regulators
(e.g. ABA)
Avoidance
Whole plant morphological, anatomical
and physiological changes
Genetic engineering
QTLs etc.
Adaptation
The ability of a genotype to survive and produce biomass relative
to other species
Current Opinion in Biotechnology
Applied biotechnology: the interacting factors in molecular and conventional breeding for plant tolerance to abiotic stress. The major types of
abiotic stress can be grouped under osmotic stress (resulting from drought and salinity, as well as freezing) and other environmental factors.
These activate specific genes that are, in turn, responsible for the expression of structural (e.g. membrane) and functional (e.g. enzymatic)
proteins, resulting in many cases in impaired metabolism, generation of reactive oxygen species (ROS), and synthesis or catabolism of several
growth regulators (the foremost being abscisic acid [ABA]). The whole gene activation machinery — if well coordinated — is manifested either in
stress tolerance (see definition) or stress avoidance. Both are responsible for plant adaptation to stress (see definition), which is the final aim of
breeding specific genotypes for actual tolerance under field conditions. Tolerance is amenable to genetic engineering of specific genes, whereas
whole-plant avoidance strategies depend on more conventional breeding schemes and quantitative trait loci (QTL) analysis.
plant breeding for abiotic stress tolerance can be achieved
only by combining traditional and molecular breeding.
Conclusions and future perspectives
Abiotic stresses, especially salinity, drought, temperature
and oxidative stress, are the primary causes of plant loss
worldwide. Therefore, plant biotechnologies aimed at
overcoming severe environmental stresses need to be
quickly and fully implemented, with intensive molecular-assisted traditional breeding and genetic engineering
being at the forefront.
In carrying out these studies on tolerance to abiotic
stress, a number of factors should be taken into consideration. Firstly, we should keep in mind that a given
tolerance-related mechanism should always be assessed
with respect to its cross-talk with other stress-related
genes/mechanisms. Secondly, most current studies use
short-term stress treatments, rather than observing the
effects of stress over longer periods — conditions that
more closely mimic the life span of most crops. As
physiological and molecular responses during short
and long exposures to stress could differ, conclusions
regarding actual tolerance, which must involve practical
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factors such as biomass, yield data and survival, cannot
always be drawn. Thirdly, cycles of stress and recovery
from stress (e.g. rehydration) are the prevalent processes
occurring under natural conditions during different seasons, and under agricultural practices such as irrigation
and salt leaching. Thus, the degree of recovery from
stress, which also has its molecular basis, is as relevant as
the response to stress.
Our limited knowledge of stress-associated metabolism is
still a major gap in our understanding of stress tolerance in
many plant species. Therefore, comprehensive profiling
of stress-associated metabolites, combined with stress
metabolomics of major crop plants will be a key factor
in molecular breeding for tolerance. Another area for
future studies will be the detailed analysis of physiological and molecular mechanisms underlying salt tolerance
in salt-tolerant model species, which will enable future
molecular dissection of salt-tolerance mechanisms in
important crop plants. It is reasonable to assume that a
thorough comparative study of the expression and function of members of the same gene families in extreme
halophytes and xerophytes will eventually assist in the
breeding of salt-tolerant crop plants.
Current Opinion in Biotechnology 2005, 16:123–132
130 Plant biotechnology
Update
Two recent papers highlight the possible role of small
HSPs (sHSPs) and LEA proteins in tolerance to
temperature and desiccation stress.
The in vivo function of sHSPs in thermoprotection was
successfully demonstrated by the constitutive expression
of anti-sHSP single-chain fragment variable (scFv) antibodies in tobacco [57]. When scFv-transgenic plants were
subjected to prolonged high temperature conditions they
became yellow and then died, suffering mainly from total
destruction of cellular membranes. The expression of the
specific scFv prevented the formation of functional heat
stress granules (HSGs), and resulted consequently in the
collapse of different cell compartments at sublethal
temperatures.
In vitro, the presence of recombinant LEA proteins from
the nematode Aphelenchus avenae (AavLEA1) and from
wheat (Em) alone did not protect citrate synthase and
lactate dehydrogenase from heat inactivation, but they
exhibited a protective effect synergistically with the
chemical chaperone trehalose. On the other hand, LEA
proteins alone showed in vitro anti-aggregation activity
upon desiccation and freezing of the enzymes, suggesting
the involvement of LEA proteins in preventing the formation of aggregates of denaturated protein during water
stress [58].
Acknowledgements
This work was supported by the European Union (grant number
QLRT-2001-00841 ‘ROST’ to A Altman).
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
of special interest
of outstanding interest
1.
Ashraf M: Breeding for salinity tolerance in plants. Crit Rev Plant
Sci 1994, 13:17-42.
2.
Wang W, Vinocur B, Altman A: Plant responses to drought,
salinity and extreme temperatures: towards genetic
engineering for stress tolerance. Planta 2003, 218:1-14.
3.
Seki M, Kamei A, Yamaguchi-Shinozaki K, Shinozaki K: Molecular
responses to drought, salinity and frost: common and
different paths for plant protection. Curr Opin Biotechnol 2003,
14:194-199.
4.
Shinozaki K, Yamaguchi-Shinozaki K, Seki M: Regulatory
network of gene expression in the drought and cold stress
responses. Curr Opin Plant Biol 2003, 6:410-417.
5.
Wang W, Vinocur B, Shoseyov O, Altman A: Role of plant heatshock proteins and molecular chaperones in the abiotic stress
response. Trends Plant Sci 2004, 9:244-252.
This comprehensive review summarizes the involvement of Hsps and
chaperones in abiotic stress responses in plants, and discusses the
cooperation among different classes and their interactions with other
stress-induced components.
6.
Park EJ, Jeknic Z, Sakamoto A, Denoma J, Yuwansiri R, Murata N,
Chen TH: Genetic engineering of glycinebetaine synthesis in
tomato protects seeds, plants, and flowers from chilling
damage. Plant J 2004, 40:474-487.
Current Opinion in Biotechnology 2005, 16:123–132
7.
Apse MP, Blumwald E: Engineering salt tolerance in plants.
Curr Opin Biotechnol 2002, 13:146-150.
8.
Rontein D, Basset G, Hanson AD: Metabolic engineering of
osmoprotectant accumulation in plants. Metab Eng 2002,
4:49-56.
9. Flowers TJ: Improving crop salt tolerance. J Exp Bot 2004,
55:307-319.
An excellent and critical review of the genetic and physiological complexity of plant salt tolerance, with special emphasis on physiological traits
relative to molecular data.
10. Zhang JZ, Creelman RA, Zhu JK: From laboratory to field. Using
information from Arabidopsis to engineer salt, cold, and
drought tolerance in crops. Plant Physiol 2004, 135:615-621.
An excellent perspective describing the abiotic stress signal transduction
pathways in Arabidopsis and their application for production of plant
tolerance to a variety of stresses.
11. Shou H, Bordallo P, Fan JB, Yeakley JM, Bibikova M, Sheen J,
Wang K: Expression of an active tobacco mitogen-activated
protein kinase kinase kinase enhances freezing tolerance
in transgenic maize. Proc Natl Acad Sci USA 2004,
101:3298-3303.
Achievements in engineering important crop plants for tolerance to
abiotic stress are limited. Here, the expression of a heterologous tobacco
MAPKKK (Nicotiana PK1) in the major cereal crop maize was studied. It
was demonstrated that constitutive expression of the Nicotiana PK1 gene
enhances freezing tolerance in transgenic maize plants that are normally
frost-sensitive
12. Qiu QS, Guo Y, Dietrich MA, Schumaker KS, Zhu JK: Regulation
of SOS1, a plasma membrane Na+/H+ exchanger in
Arabidopsis thaliana, by SOS2 and SOS3. Proc Natl Acad Sci
USA 2002, 99:8436-8441.
13. Thiery L, Leprince AS, Lefebvre D, Ghars MA, Debarbieux E,
Savoure A: Phospholipase D is a negative regulator of proline
biosynthesis in Arabidopsis thaliana. J Biol Chem 2004,
279:14812-14818.
14. Lee S, Lee EJ, Yang EJ, Lee JE, Park AR, Song WH, Park OK:
Proteomic identification of annexins, calcium-dependent
membrane binding proteins that mediate osmotic stress and
abscisic acid signal transduction in Arabidopsis. Plant Cell
2004, 16:1378-1391.
Using comparative proteomic analysis a stress-responsive Ca2+-dependent membrane-binding protein, designated as annexin 1, was identified.
The annexin 1 gene transcript was constitutively expressed, while protein
expression was upregulated by stress and the protein translocated from
the cytosol to the membrane.
15. Sung DY, Guy CL: Physiological and molecular assessment of
altered expression of Hsc70-1 in Arabidopsis. Evidence for
pleiotropic consequences. Plant Physiol 2003, 132:979-987.
16. Sun W, Van Montagu M, Verbruggen N: Small heat shock
proteins and stress tolerance in plants. Biochim Biophys Acta
2002, 1577:1-9.
17. Kore-eda S, Cushman MA, Akselrod I, Bufford D, Fredrickson M,
Clark E, Cushman JC: Transcript profiling of salinity stress
responses by large-scale expressed sequence tag analysis in
Mesembryanthemum crystallinum. Gene 2004, 27:83-92.
18. Wang D, Luthe DS: Heat sensitivity in a bentgrass variant.
Failure to accumulate a chloroplast heat shock protein
isoform implicated in heat tolerance. Plant Physiol 2003,
133:319-327.
19. Yamada A, Sekiguchi M, Mimura T, Ozeki Y: The role of plant
CCTa in salt- and osmotic-stress tolerance. Plant Cell Physiol
2002, 43:1043-1048.
20. Katiyar-Agarwal S, Agarwal M, Grover A: Heat-tolerant basmati
rice engineered by over-expression of hsp101. Plant Mol Biol
2003, 51:677-686.
21. Villalobos MA, Bartels D, Iturriaga G: Stress tolerance and
glucose insensitive phenotypes in Arabidopsis
overexpressing the CpMYB10 transcription factor gene.
Plant Physiol 2004, 135:309-324.
22. Chandra Babu R, Zhang J, Blum A, Hod THD, Wue R, Nguyen HT:
HVA1, a LEA gene from barley confers dehydration tolerance
www.sciencedirect.com
Engineering plant tolerance to abiotic stress Vinocur and Altman 131
in transgenic rice (Oryza sativa L.) via cell membrane
protection. Plant Sci 2004, 166:855-862.
23. Mittler R: Oxidative stress, antioxidants and stress tolerance.
Trends Plant Sci 2002, 7:405-410.
24. Yoshimura K, Miyao K, Gaber A, Takeda T, Kanaboshi H,
Miyasaka H, Shigeoka S: Enhancement of stress tolerance
in transgenic tobacco plants overexpressing Chlamydomonas
glutathione peroxidase in chloroplasts or cytosol. Plant J 2004,
37:21-33.
41. Jang IC, Oh SJ, Seo JS, Choi WB, Song SI, Kim CH, Kim YS,
Seo HS, Choi YD, Nahm BH et al.: Expression of a bifunctional
fusion of the Escherichia coli genes for trehalose-6-phosphate
synthase and trehalose-6-phosphate phosphatase in
transgenic rice plants increases trehalose accumulation
and abiotic stress tolerance without stunting growth.
Plant Physiol 2003, 131:516-524.
A recent study on the stress-inducible overexpression of the bifunctional
trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase fusion gene in transgenic rice. This work is likely to offer novel
strategies for improving abiotic stress tolerance in crop plants.
25. Sunkar R, Bartels D, Kirch HH: Overexpression of a stressinducible aldehyde dehydrogenase gene from Arabidopsis
thaliana in transgenic plants improves stress tolerance.
Plant J 2003, 35:452-464.
42. Penna S: Building stress tolerance through over-producing
trehalose in transgenic plants. Trends Plant Sci 2003,
8:355-357.
26. Kirch HH, Bartels D, Wei Y, Schnable P, Wood A: The aldehyde
dehydrogenase gene superfamily of Arabidopsis thaliana.
Trends Plant Sci 2004, 9:371-377.
43. Abebe T, Guenzi AC, Martin B, Cushman JC: Tolerance of
mannitol-accumulating transgenic wheat to water stress and
salinity. Plant Physiol 2003, 131:1748-1755.
27. Capell T, Christou P: Progress in plant metabolic engineering.
Curr Opin Biotechnol 2004, 15:148-154.
44. Rizhsky L, Liang H, Shuman J, Shulaev V, Davletova S, Mittler R:
When defense pathways collide. The response of Arabidopsis
to a combination of drought and heat stress. Plant Physiol 2004,
134:1683-1696.
Metabolic profiling of plants subjected to drought, heat stress or a
combination of the two revealed that plants subject to a combination
of drought and heat stress accumulated sucrose and other sugars such
as maltose and glucose.
28. Konstantinova T, Parvanova D, Atanassov A, Djilianov D: Freezing
tolerant tobacco, transformed to accumulate
osmoprotectants. Plant Sci 2002, 163:157-164.
29. Mani S, van de Cotte B, van Montagu M, Verbruggen N: Altered
levels of proline dehydrogenase cause hypersensitivity to
proline and its analogs in Arabidopsis. Plant Physiol 2002,
128:73-83.
30. Nanjo T, Fujita M, Seki M, Kato T, Tabata S, Shinozaki K:
Toxicity of free proline revealed in an Arabidopsis T-DNAtagged mutant deficient in proline dehydrogenase. Plant Cell
Physiol 2003, 44:541-548.
31. Maggio A, Miyazaki S, Veronese P, Fujita T, Ibeas JI, Damsz B,
Narasimhan ML, Hasegawa PM, Joly RJ, Bressan RA: Does
proline accumulation play an active role in stress-induced
growth reduction? Plant J 2002, 31:699-712.
32. Khedr AH, Abbas MA, Wahid AA, Quick WP, Abogadallah GM:
Proline induces the expression of salt-stress-responsive
proteins and may improve the adaptation of Pancratium
maritimum L. to salt-stress. J Exp Bot 2003, 54:2553-2562.
33. Li QL, Gao XR, Yu XH, Wang XZ, An LJ: Molecular cloning and
characterization of betaine aldehyde dehydrogenase gene
from Suaeda liaotungensis and its use in improved tolerance
to salinity in transgenic tobacco. Biotechnol Lett 2003,
25:1431-1436.
34. Mohanty A, Kathuria H, Ferjani A, Sakamoto A, Mohanty P,
Murata N, Tyagi AK: Transgenics of an elite indica rice variety
Pusa Basmati 1 harbouring the codA gene are highly tolerant
to salt stress. Theor Appl Genet 2002, 106:51-57.
35. Yilmaz JL, Bulow L: Enhanced stress tolerance in Escherichia
coli and Nicotiana tabacum expressing a betaine aldehyde
dehydrogenase/choline dehydrogenase fusion protein.
Biotechnol Prog 2002, 18:1176-1182.
36. Friedman R, Altman A, Levin N: The effect of salt stress on
polyamine biosynthesis and content in mung bean plants and
in halophytes. Physiol Plant 1989, 76:295-302.
37. Capell T, Bassie L, Christou P: Modulation of the polyamine
biosynthetic pathway in transgenic rice confers tolerance to
drought stress. Proc Natl Acad Sci USA 2004, 101:9909-9914.
38. Kasukabe Y, He L, Nada K, Misawa S, Ihara I, Tachibana S:
Overexpression of spermidine synthase enhances tolerance
to multiple environmental stresses and up-regulates the
expression of various stress-regulated genes in transgenic
Arabidopsis thaliana. Plant Cell Physiol 2004, 45:712-722.
39. Silva JMd, Arrabaça MC: Contributions of soluble
carbohydrates to the osmotic adjustment in the C4 grass
Setaria sphacelata: a comparison between rapidly and slowly
imposed water stress. J Plant Physiol 2004, 161:551-555.
40. Garg AK, Kim JK, Owens TG, Ranwala AP, Choi YD, Kochian LV,
Wu RJ: Trehalose accumulation in rice plants confers high
tolerance levels to different abiotic stresses. Proc Natl Acad Sci
USA 2002, 99:15898-15903.
www.sciencedirect.com
45. Bartels D, Salamini F: Desiccation tolerance in the resurrection
plant Craterostigma plantagineum. A contribution to the study
of drought tolerance at the molecular level. Plant Physiol 2001,
127:1346-1353.
46. Hilbricht T, Salamini F, Bartels D: CpR18, a novel SAP-domain
plant transcription factor, binds to a promoter region
necessary for ABA mediated expression of the CDeT27-45
gene from the resurrection plant Craterostigma plantagineum
Hochst. Plant J 2002, 3:293-303.
47. Deng X, Phillips J, Meijer AH, Salamini F, Bartels D:
Characterization of five novel dehydration-responsive
homeodomain leucine zipper genes from the resurrection
plant Craterostigma plantagineum. Plant Mol Biol 2002,
49:601-610.
48. Hurst AC, Grams TEE, Ratajczak R: Effects of salinity, high
irradiance, ozone, and ethylene on mode of photosynthesis,
oxidative stress and oxidative damage in the C3/CAM
intermediate plant Mesembryanthemum crystallinum L.
Plant Cell Environ 2004, 27:187-197.
49. Su H, Balderas E, Vera-Estrella R, Golldack D, Quigley F,
Zhao C, Pantoja O, Bohnert HJ: Expression of the cation
transporter McHKT1 in a halophyte. Plant Mol Biol 2003,
52:967-980.
50. Vera-Estrella R, Barkla BJ, Bohnert HJ, Pantoja O: Novel
regulation of aquaporins during osmotic stress. Plant Physiol
2004, 135:2318-2329.
51. Inan G, Zhang Q, Li P, Wang Z, Cao Z, Zhang H, Zhang C,
Quist TM, Goodwin SM, Zhu J et al.: Salt cress. A halophyte
and cryophyte Arabidopsis relative model system and its
applicability to molecular genetic analyses of growth and
development of extremophiles. Plant Physiol 2004,
135:1718-1737.
The authors highlight the use of salt cress (Thellungiella halophyla) as a
new model plant for salt stress tolerance. Salt cress is a winter crucifer
annual halophyte and closely related to Arabidopsis; both salt cress and
Arabidopsis genomes share a high percentage of nucleotide similarity.
Microarray analysis of salt cress RNA can be successfully carried out in
Arabidopsis whole-genome arrays.
52. Taji T, Seki M, Satou M, Sakurai T, Kobayashi M, Ishiyama K,
Narusaka Y, Narusaka M, Zhu JK, Shinozaki K: Comparative
genomics in salt tolerance between Arabidopsis and
Arabidopsis-related halophyte salt cress using Arabidopsis
microarray. Plant Physiol 2004, 135:1697-1709.
53. Volkov V, Wang B, Dominy PJ, Fricke W, Amtmann A:
Thellungiella halophila, a salt-tolerant relative of Arabidopsis
thaliana, possesses effective mechanisms to discriminate
between potassium and sodium. Plant Cell Environ 2003,
27:1-14.
Current Opinion in Biotechnology 2005, 16:123–132
132 Plant biotechnology
54. Lin HX, Zhu MZ, Yano M, Gao JP, Liang ZW, Su WA, Hu XH,
Ren ZH, Chao DY: Mapping of quantitative trait loci controlling
low-temperature germinability in rice (Oryza sativa L.). Theor
Appl Genet 2004, 108:794-799.
55. Saranga Y, Jiang CX, Wright RJ, Yakir D, Paterson AH: Genetic
dissection of cotton physiological responses to arid
conditions and their inter-relationships with productivity.
Plant Cell Environ 2004, 27:263-277.
56. Tsarouhas V, Gullberg U, Lagercrantz U: Mapping of
quantitative trait loci (QTLs) affecting autumn freezing
Current Opinion in Biotechnology 2005, 16:123–132
resistance and phenology in Salix. Theor Appl 2004,
108:1335-1342.
57. Miroshnichenko S, Tripp J, Nieden UZ, Neumann D, Conrad U,
Manteuffel R: Immunomodulation of function of small heat
shock proteins prevents their assembly into heat stress
granules and results in cell death at sublethal temperatures.
Plant J 2005, 41:269-281.
58. Goyal K, Walton LJ, Tunnacliffe A: LEA proteins prevent
protein aggregation due to water stress. Biochem J 2005:
in press.
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