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Review

Effects of brassinosteroids on the plant responses to environmental stresses

abstract

Brassinosteroids are found in a wide range of organisms from lower to higher plants. They are
steroidal plant hormones implicated in the promotion of plant growth and development.
Brassinosteroid metabolism has long been known to be altered in plants responding to abiotic
stresses and to undergo profound changes in plants interacting with bacterial, fungal and viral
pathogens. This review describes the role of brassinosteroids in response to various kinds of
stresses via activation of different mechanisms.

1. Introduction The brassinosteroidsdbrassinolide (BL) and castasterone (CS)doccur ubiquitously in


the plant kingdom. The occurrence of brassinosteroids (BRs) has been demonstrated in almost
every part of plants, such as pollen, flower buds, fruits, seeds, vascular cambium, leaves, shoots
and roots. These steroidal compounds occur in free form and conjugated to sugars and fatty acids.
To this date, about 70 BRs have been isolated from plants [10]. BRs are required for normal
development of plants. Works on higher plants suggest that they play a critical role in a range of
developmental processes, e.g. stem and root growth, floral initiation, and the development of
flowers and fruits [27,54]. BRs have also been implicated in plant responses to abiotic stress.
Despite the correlation between abiotic stress and BR levels in higher plants, the physiological
rationale for such alteration in BR levels is still not known. These hormones are also known to
change in plants responding to biotic stress, e.g. pathogen infection [40].

2. Plant response to stresses

Plants respond to abiotic and biotic factors in environment. These include heavy metals action,
wounding, drought, high salt, and changes in temperature and light, and pathogen and pest
attack. Abiotic stress leads to a morphological, physiological, biochemical and molecular changes.
Drought, salinity, extreme temperatures and oxidative stress are often interconnected, and may
induce similar cellular damage. For example, drought and/or salinization are manifested primarily
as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cells.
Oxidative stress, which frequently accompanies high temperature, salinity, or drought stress, may
cause denaturation of functional and structural proteins. As a consequence, these diverse
environmental stresses often activate similar cell signalling pathways and cellular responses, such
as the production of stress proteins, upregulation of antioxidants and accumulation of compatible
solutes. Biochemical adaptation in plants involves various changes in the biochemistry of the cell.
These changes include the evolution of new metabolic pathways, the accumulation of low
molecular weight metabolites, the synthesis of special proteins, detoxification mechanisms and
changes in phytohormone levels. Adaptation represents the ability of a living organism to fit into a
changing environment, at the same time improving its chances of survival and reproducing itself
[21,63].
3. Brassinosteroid-mediated detoxification systems

3.1. Cross-talk between brassinosteroids and other plant hormones

Coordinated regulation of plant development in response to the environment requires a cross-talk


between hormones and the pathways initiated by the external cues. Plant responses to stress can
be viewed as being orchestrated through a network that integrates signalling pathways
characterized by the production of ethylene and jasmonic acid (JA), salicylic acid (SA). The
identified regulatory steps in the network involve transcription, protein– protein interaction and
targeted protein destruction. In plants, the mitogen-activated protein (MAP) kinase cascade plays
a key role in various abiotic and biotic stress responses and in phytohormones responses that
include reactive oxygen species (ROS) signalling (Fig. 1) [21]. Molecular studies present the notion
that the cross-talk between BRs and other phytohormones exists. It includes alternation in the
expression of hormone biosynthetic genes and/or signalling intermediates. Studies of such
interactions have examined changes in endogenous levels of other hormones, described
synergistic effects with exogenous BR treatments. BRs increased ethylene production in mung
bean epicotyl segments [7]. Also, BRs have an additive effect with gibberellins (GA) [44] or a
synergistic effect with auxin on stem segment elongation [15, 37]. In intact Arabidopsis thaliana
seedlings, co-application of BRs and GA, or BRs and auxin, resulted in a synergistic increase in
hypocotyl elongation [66]. An interaction of ABA and BRs in cell elongation has been shown in
Arabidopsis thaliana bee1, bee2, bee3 mutants [19]. These mutants were less responsive to
exogenous brassinolide (BL) in hypocotyl growth assays, and the triple mutant phenotype was
similar to bri1 in terms of reduced hypocotyl growth and floral organ size. Expression of proteins
named BR Enhanced Expression (BEE1, BEE2 and BEE3) was repressed by ABA treatment. ABA is a
known antagonist of BR signalling. Reduced ABA response in plants overexpressing BEE1 suggests
that BEE proteins may function as signalling intermediates in multiple pathways. It is possible that
BR plays a role in down-regulating some responses to ABA or when BRs is lacking may amplify or
modify the ABA signal. However, another study suggested that ABA and BR both enhance drought
tolerance in sorghum (Sorghum vulgare) [75]. One of the signal molecules which integrated in the
regulation of stress response and plant development is jasmonic acid (JA). The biosynthesis of JA is
initiated by oxygenation of a-linolenic acid to 13-hydroperoxy linolenic acid through the action of a
lipoxygenase. On the other hand, 13-hydroperoxy linolenic acid is metabolized by allene oxide
synthase (AOS). Subsequently, 12-oxophytodienoic acid (OPDA) is formed by allene oxide cyclase
(AOC). After the reduction of a double bond in the cyclopentenone ring of OPDA to
12-oxo-phytoenoic acid (OPC8:0) by 12-oxo-phytodienoic acid reductase (OPR). Thus, JA is
synthesized from OPDA by the intermediates OPC8:0, OPC6:0 and OPC4:0 [57]. The expression of
the OPR3 gene is induced by BRs, JA, and by a variety of stimuli like UVlight, touch, wind or
wounding. As OPR3 expression is increased through treatment with BRs, it indicates a potential
link between BR action and JA biosynthesis. BRs may thus influence the stress responses of plants
through stimulation of JA biosynthesis [46,56].

3.2. Response to oxidative stress


Reactive oxygen species (ROS) play a key role in plant growth, development, and interaction with
biotic and abiotic stresses. ROS have also been implicated as important regulatory and signalling
elements in a variety of cellular processes. ROS are constantly produced during the course of
photosynthesis and respiration, whereas redox homeostasis in the cell is tightly controlled by
redundant protective mechanisms. Disruption of these protective mechanisms can cause oxidative
stress, leading to oxidative damage and death of cell. Although, under normal growth conditions,
the production of ROS in cells is very low, many stresses that disrupt the cellular homeostasis of
cells enhance the production of ROS. These stresses include drought stress and desiccation, salt
stress, chilling, heat shock, heavy metals, ultraviolet radiation, ozone, mechanical stress, nutrient
deprivation, pathogen attack and high light stress. When plants are subjected to stresses, a variety
of ROS are generated, such as superoxide radical (O2 $), hydrogen peroxide (H2O2) and hydroxyl
radical ($ OH) [22]. BRs, which play an essential role in plant growth and development, have been
implicated in many physiological responses [54]. However, little is known about the role of BRs in
the plant response to oxidative stress. It was shown that exogenous application of BRs modified
antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and
ascorbate peroxidase and non-enzymatic antioxidants, such as ascorbic acid, tocopherols,
carotenoids, glutathione, etc. in plants under different stress conditions. Vitamins C, E, and
glutathione react directly or via enzyme catalysis with $ OH, H2O2 or O2 $, while carotenes
directly operate as effective quenchers of reactive intermediary forms of oxygen [42,49,50,69].
When maize (Zea mays) seedlings treated with BL were subjected to water stress, the activities of
superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), as well as ascorbic acid
and carotenoids contents increased [42]. On the other hand, BRs enhanced the activity of CAT and
reduced the activities of peroxidase and ascorbic acid oxidase under osmotic stress conditions in
sorghum (Sorghum vulgare) [69]. Rice seedlings exposed to saline stress and treated with BR
showed a significant increase in the activities of CAT, SOD and glutathione reductase (GR) and a
slight increase in APX [49]. A comparison between salt-sensitive and salt-resistant varieties of rice
treated with 24-epiBL was also reported [50]. 24-epiBL treatment, at least in part, improved the
tolerance of salt-sensitive seedlings to short-term salt stress. The difference in alteration of
antioxidant enzyme activities in salt-sensitive rice cultivar may suggest that the higher salt stress
resistance in sensitive seedlings induced by 24-epiBL may be due in part to a maintained higher
activity of APX under salt stress conditions. Algae Chlorella vulgaris respond to heavy metals
(cadmium, copper and lead) by induction of several antioxidants, including several enzymatic
systems, and the synthesis of low molecular weight compounds. Treatment with BL was effective
in increasing the activity of antioxidant enzymes (CAT, GR, and APX) and the content of ascorbic
acid, carotenoids and glutathione (Bajguz, unpublished data). The influence of 24-epiBL on some
enzymatic antioxidants in tomato leaf disc under high (40 C) temperature was reported [45]. BR
increased the activity of CAT, peroxidase and SOD in respond to high temperature in tomato
leaves. It has been shown that the det2 Arabidopsis mutant, which is blocked in the biosynthetic
pathway of BRs [13], had significantly thicker leaves with thickened cuticle layers and cell walls in
the epidermal and mesophyll layers, increased stomatal density and compacted leaf structure with
less intercellular space than the wild-type when grown under normal oxygen conditions. Addition
of BL to the growth medium resulted in leaves that were more similar in morphology to those of
Columbia. Interestingly, the det2 mutant was also found to be insensitive to the dwarfing effects
of very low O2 (2.5 kPa) and furthermore did not show the purpling stress response of wild-type of
Arabidopsis. BRs could be as possible mediators of particular aspects of the developmental
response to hypoxia, a conclusion supported by the fact that BRs require molecular oxygen at
several steps in their biosynthesis [51]. It has been also demonstrated that ATPA2 and ATP24a
genes encoding peroxidases were constitutively upregulated in the det2 Arabidopsis mutant [24].
Furthermore, the oxidative stress-related genes encoding monodehydroascorbate reductase and
thioredoxin, the cold and drought stress response genes COR47 and COR78, and the heat
stress-related genes HSP83, HSP70, HSF3, Hsc70-3 and Hsc70- G7 have been identified by
microarray analysis of either BR-defi- cient or BR-treated plants [47]. The enhanced oxidative
stress resistance in det2 plants was correlated with a constitutive increase in SOD activity and
increased transcript levels of the defence gene CAT. Therefore, a possible explanation for the fact
that the det2 mutant exhibited an enhanced oxidative stress resistance is that the long-term BR
deficiency in the det2 mutant results in a constant in vivo physiological stress that, in turn,
activates the constitutive expression of some defence genes and, consequently, the activities of
related enzymes. If this is the case, it may suggest that endogenous BRs in wild-type plants
somehow act to repress the transcription or post-transcription activities of the defence genes to
ensure the normal growth and development of plants. However, it is still unclear whether BRs
directly or indirectly modulate the responses of plants to oxidative stress [12].

3.3. Response to osmotic stress

Drought, salinity and freeze-induced dehydration constitute direct osmotic stresses; whereas
chilling and hypoxia can indirectly cause osmotic stress via effects on water uptake and loss. Plants
respond to dehydration and low temperature with a number of physiological and developmental
changes. Plants have evolved a high capacity to synthesize and accumulate non-toxic solutes
(osmoprotectants), for example proline, glycine betaine, mannitol, and trehalose. Drought and
high salinity cause plants to produce high levels of abscisic acid (ABA); exogenous application of
ABA also induces a number of genes that respond to dehydration and cold stress. Nevertheless,
the role of ABA in low-temperatureresponsive gene expression is not clear. Several reports have
described genes that are induced by dehydration and low temperature. It is likely, therefore, that
both ABA-independent and ABA-dependent signal transduction cascades exist [67]. Proline is
accumulated in many plant species in response to osmotic stress, which is stimulated by drought,
salt, cold and ABA. The accumulation of proline is dependent on the expression of D1 -
pyrroline-5-carboxylate synthase (P5CS) and proline dehydrogenase (PDH). These enzymes
catalyze the rate-limiting steps of proline biosynthesis and degradation, respectively. Stimulation
of proline synthesis by ABA and salt stress is correlated with a striking activation of P5CS1
expression. By contrast, P5CS2 is only weakly induced, whereas PDH is inhibited to different extent
by ABA and salt stress in light-grown Arabidopsis thaliana. However, P5CS2 is also down-regulated,
whereas PDH expression is significantly enhanced in dark-grown shoots of Arabidopsis. In
comparison, P5CS1 expression is inhibited in dark-grown plants. The inhibitory effect of dark
adaptation on P5CS1 is mimicked by brassinolide (BL). However, BL fails to stimulate PDH, and
inhibits P5CS2 only in shoots. Proline accumulation and induction of P5CS1 transcription are
simultaneously enhanced in the ABA-hypersensitive prl1 and BR-deficient det2 mutants, whereas
P5CS2 shows enhanced induction by ABA and salt only in the det2 mutant. Salt stress is expected
to stimulate an ABA-signalling pathway, which requires the ABA1 and ABI1 functions and triggers
transcriptional activation of P5CS1 by an ABA-responsive promoter element. The fact, that both
ABA and salt induction of P5CS1 transcription is inhibited by BL in light-grown plants suggests that
BRs may negatively regulate this common salt and ABA response pathway. Alternatively, BL may
inhibit the light or sugar (or both) regulated maintenance of basal P5CS1 transcription, which is
essential for further induction by salt and ABA [1]. The effect of BRs on changes in root nodulation
of Phaseolus vulgaris and contents of endogenous ABA and cytokinin transzeatin riboside (ZR) has
been reported [68]. BRs in the unstressed plants increased root nodulation, ZR content and also
ameliorated their stress induced decline in the nodulated roots. However, ABA contents in the
nodules of control or stressed plants were not altered by BRs treatment. Water stress induced
decline in root nodulation is associated with increase in ABA and decline in cytokinins contents in
the nodulated roots. BRs have the potential to improve root nodulation and pod yield in the
irrigated and water stressed plants; an effect that could be mediated through an influence on
cytokinin content in the nodulated roots of Phaseolus vulgaris. BRs application also resulted in
enhancement of seedling growth, which was evident in terms of seedling length, seedling fresh
and dry weights of all the three varieties of sorghum (Sorghum vulgare) viz. CSH-14 and ICSV-745
(susceptible to water stress) and M-35-1 (resistant to water stress) under osmotic stress [69].
Similar results have been shown in sugar-beet plants under drought stress, in which a reduction of
taproot weight was correlated to stress severity. Treatment with BR fully compensated for the
reduction in biomass caused by mild drought stress. On the other hand, increases in biomass was
correlated with increases in acid invertase activity in young leaves, which likely provided more
assimilates to the plant due to their larger sizes [58]. Furthermore, osmotic stress resulted in
considerable reduction in the protein contents in all the three varieties of sorghum. However, BRs
not only restored but also stimulated the level of protein and free proline [69]. HomoBL also had a
stimulatory effect on the growth of droughttolerant (C306) and drought-susceptible (HD2329)
wheat (Triticum aestivum) varieties under stress conditions. Application of homoBL resulted in
increased relative water content, nitrate reductase activity, chlorophyll content and
photosynthesis under both conditions. It also improved membrane stability (lower injury). These
beneficial effects resulted in higher leaf area, biomass production, grain yield and yield related
parameters in the treated plants. However, homoBL showed a higher activity in droughttolerant
wheat variety under water stress conditions. Increased water uptake and membrane stability and
higher carbon dioxide and nitrogen assimilation rates in BR-treated plants under stress were
correlated with BR-induced drought tolerance [53]. BRs also improved the protoplasmic drought
tolerance of suspended cells from hydrated leaves of Sporobolus stapfianus. Hydrated leaves of
the resurrection grass are not desiccation tolerant, but tolerance can be induced by drought stress
[23].

3.4. Response to heavy metals stress

Plants have a remarkable ability to take up and accumulate heavy metals from their external, for
example aquatic environment. Metal contamination of the aquatic environment occurs as a result
of human activities and affects organisms at the biochemical, cellular, community and population
level. Aquatic plants are primary producers of most aquatic food chains and account for much of
the production base of freshwater and marine ecosystems. They have been used to remove heavy
metals from aquatic environments since they have a high capability for accumulating dissolved
metals without dying. High concentrations of all metals, including those essential for growth and
metabolism, exert toxic effects on the metabolic pathways of plants. Toxicity mechanisms include
the blocking of functional groups of important molecules, e.g. enzymes, polynucleotides, transport
systems for essential nutrients and ions, displacement and/or substitution of essential ions from
cellular sites, denaturation and inactivation of enzymes, and disruption of cell and organellar
membrane integrity. In addition, metals are also pointed to exert toxic effects through free radical
formation, the phenomenon found to be least explored [63]. Heavy metal toxicity can elicit a
variety of adaptive responses in plants. A ubiquitous mechanism for heavy metal detoxification is
the chelation of the metal ion by a ligand. Such ligands include organic acids, amino acids, peptides
and polypeptides. Peptide ligands include the phytochelatins (PC), small gene-encoded,
cysteine-rich polypeptides. They are heavy metal-binding peptides derived from glutathione and
have the general structure (g-GluCys)n-Gly, where n has been reported as being as high as 11, but
is generally in the range of 2–5. PC detoxifies intracellular metals by binding them through thiolate
coordination [14]. BRs stimulated the synthesis of PC in Chlorella vulgaris cells treated with lead.
The stimulatory activity of BRs on PC synthesis was arranged in the following order: brassinolide
(BL) > 24-epiBL > 28-homoBL > castasterone (CS) > 24-epiCS > 28-homoCS [9]. The cultures of
Chlorella vulgaris treated with BRs and heavy metals show a lower bioaccumulation of heavy
metals than the cultures treated with metals alone. The inhibitory effect of BRs mixed with
different heavy metals on their accumulation was arranged in the following order: zinc > cadmium
> lead > copper. A stimulatory effect of BRs after blocking the accumulation of heavy metals on the
growth and development of Chlorella vulgaris occurs. Application of BRs to Chlorella vulgaris
cultures reduced the impact of heavy metals stress on growth, prevented chlorophyll, sugar and
protein loss and increased PC synthesis. Concentration-dependent stimulation was observed with
increasing concentration of BR and decreasing concentration of heavy metals [8,9]. BRs also
reduced the content of cadmium in the seedlings of winter rape [35] and copper in Indian mustard
[61]. BR eliminates the toxic effect of cadmium on photochemical pathways in rape cotyledons,
mainly by diminishing the damage in reaction centres and O2 evolving complexes as well as
maintaining efficient photosynthetic electron transport [35]. The accumulation of heavy metals
(cadmium, copper, lead and zinc) under the influence of BR has been studied for different
agricultural plants such as barley, tomato, radish and sugar beet. It was found that the application
of 24-epiBL signifi- cantly reduced the metal absorption; for example, the content of lead in beet
roots was more than 50% lower than in the control culture [38]. Moreover, Bilkisu et al. [11]
reported that brassinolide (BL) during aluminium-related stress stimulated growth in Phaseolus
aureus. It was shown that changes in the ions/metal content were influenced by 24-epiBL and
dependent on the stage of plant development when the seeds were treated. The application of
BRs also improved the performance of mustard [29], chickpea [26] subjected to cadmium stress
and also of mungbean [3] and mustard [2] to aluminium and nickel, respectively. Hasan et al. [26]
reported that BRs enhanced activity of the antioxidant enzymes (catalase, peroxidase and
superoxidase) and proline content. It has been found a significant correlation with the degree of
improvement, which was measured in terms of nodulation, nitrogen fixation, pigment
composition, carbonic anhydrase and nitrate reductase activities. A similar pattern of response
together with an elevation in the photosynthesis was observed in the plants of mustard exposed
to cadmium through nutrient medium [29]. The foliar spray of either with 24-epiBL or 28-homoBL
significantly enhanced the growth, photosynthesis, antioxidant enzymes and proline content in
aluminium stressed mungbean plants [3]. The activities of the enzymes catalase, peroxidase,
carbonic anhydrase and nitrate reductase also exhibited a significant enhancement in mustard
plants grown under nickel stress [2]. These plants also exhibited an elevation in the relative water
content and photosynthetic performance. Ali et al. [5] also reported that 24-epiBL enhanced the
level of antioxidant system (superoxide dismutase, catalase, peroxidase and glutathione reductase
and proline), both under stress and stress-free conditions. The influence of 24-epiBL on the
antioxidant system was more pronounced under a stress situation, suggesting that the elevated
level of antioxidant system, at least in part, increased the tolerance of mustard plants to saline
and/or nickel stress, and thus protected the photosynthetic machinery and the plant growth. One
of mechanisms for the metal removal is biosorption. The mechanisms of biosorption are generally
based on physico-chemical interactions between metal ions and the functional groups present on
the cell surface, such as electrostatic interactions, ion exchange and metal ion chelation or
complexation. Functional groups most commonly implicated in such interactions include
carboxylate, hydroxyl, amine and phosphoryl groups present within cell wall components such as
polysaccharides, lipids and proteins. The binding process is independent of metabolism and hence,
of a physical nature. It is also usually rapid and reversible. In addition, biosorption can be affected
by pH and the presence of other ions in the medium which may precipitate heavy metals as
insoluble salts, but it is unaffected by metabolic inhibitors or light/ dark cycles [71]. Water pH is an
important factor directly affecting the toxicity of metals in algae, for example unicellular Chlorella
sp. It is known that heavy metal toxicity decreases with decreasing pH. Acidity or alkalinity of the
medium can, in turn, moderate the toxicity of heavy metals (copper, lead, cadmium or zinc). Lower
pH may increase the bioavailability of metal ions resulting in increased toxicity. BR-induced
Chlorella vulgaris growth stimulation depends at least partly on the decrease of pH in the wall
space, and thus on acid-induced wall loosening. The effects of BRs on proton secretion are
associated with an early hyperpolarization of the transmembrane electrical potential. BR-induced
proton excretion is also stimulated by the presence of Kþ in the medium [8].

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