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Plant Physiology and Biochemistry 47 (2009) 1–8

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Plant Physiology and Biochemistry

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Review

Effects of brassinosteroids on the plant responses to environmental stresses

Andrzej Bajguz*, Shamsul HayatDepartment of Plant Biochemistry, Institute of Biology, University of Bialystok, Swierkowa 20B, 15-950 Bialystok, PolandDepartment of Botany, Plant Physiology & Biochemistry Division, Aligarh Muslim University, Aligarh 202 002, India

a r t i c l e i n f o

Article history:Received 25 September 2008Accepted 1 October 2008Available online 17 October 2008

Keywords:BrassinosteroidsHeavy metals stressOsmotic stressOxidative stressPathogen stresssaline stressThermal stress

* Corresponding author. Tel.: þ48 85 745 7334; faxE-mail addresses: abajguz@uwb.edu.pl (A. Bajguz),

0981-9428/$ – see front matter � 2008 Elsevier Masdoi:10.1016/j.plaphy.2008.10.002

a b s t r a c t

Brassinosteroids are found in a wide range of organisms from lower to higher plants. They are steroidalplant hormones implicated in the promotion of plant growth and development. Brassinosteroidmetabolism has long been known to be altered in plants responding to abiotic stresses and to undergoprofound changes in plants interacting with bacterial, fungal and viral pathogens. This review describesthe role of brassinosteroids in response to various kinds of stresses via activation of differentmechanisms.

� 2008 Elsevier Masson SAS. All rights reserved.

1. Introduction

The brassinosteroidsdbrassinolide (BL) and castasterone(CS)doccur ubiquitously in the plant kingdom. The occurrence ofbrassinosteroids (BRs) has been demonstrated in almost every partof plants, such as pollen, flower buds, fruits, seeds, vascularcambium, leaves, shoots and roots. These steroidal compoundsoccur in free form and conjugated to sugars and fatty acids. To thisdate, about 70 BRs have been isolated from plants [10]. BRs arerequired for normal development of plants. Works on higher plantssuggest that they play a critical role in a range of developmentalprocesses, e.g. stem and root growth, floral initiation, and thedevelopment of flowers and fruits [27,54]. BRs have also beenimplicated in plant responses to abiotic stress. Despite the corre-lation between abiotic stress and BR levels in higher plants, thephysiological rationale for such alteration in BR levels is still notknown. These hormones are also known to change in plantsresponding 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 pestattack. Abiotic stress leads to a morphological, physiological,

: þ48 85 745 7302.shayat@lycos.com (S. Hayat).

son SAS. All rights reserved.

biochemical and molecular changes. Drought, salinity, extremetemperatures and oxidative stress are often interconnected, andmay induce similar cellular damage. For example, drought and/orsalinization are manifested primarily as osmotic stress, resulting inthe disruption of homeostasis and ion distribution in the cells.Oxidative stress, which frequently accompanies high temperature,salinity, or drought stress, may cause denaturation of functionaland structural proteins. As a consequence, these diverse environ-mental stresses often activate similar cell signalling pathways andcellular responses, such as the production of stress proteins, up-regulation of antioxidants and accumulation of compatible solutes.Biochemical adaptation in plants involves various changes in thebiochemistry of the cell. These changes include the evolution ofnew metabolic pathways, the accumulation of low molecularweight metabolites, the synthesis of special proteins, detoxificationmechanisms and changes in phytohormone levels. Adaptationrepresents the ability of a living organism to fit into a changingenvironment, at the same time improving its chances of survivaland 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 theenvironment requires a cross-talk between hormones and thepathways initiated by the external cues. Plant responses to stresscan be viewed as being orchestrated through a network that inte-grates signalling pathways characterized by the production of

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ethylene and jasmonic acid (JA), salicylic acid (SA). The identifiedregulatory steps in the network involve transcription, protein–protein interaction and targeted protein destruction. In plants, themitogen-activated protein (MAP) kinase cascade plays a key role invarious abiotic and biotic stress responses and in phytohormonesresponses that include reactive oxygen species (ROS) signalling(Fig. 1) [21]. Molecular studies present the notion that the cross-talkbetween BRs and other phytohormones exists. It includes alterna-tion in the expression of hormone biosynthetic genes and/or sig-nalling intermediates. Studies of such interactions have examinedchanges in endogenous levels of other hormones, describedsynergistic effects with exogenous BR treatments. BRs increasedethylene production in mung bean epicotyl segments [7]. Also, BRshave an additive effect with gibberellins (GA) [44] or a synergisticeffect with auxin on stem segment elongation [15, 37]. In intactArabidopsis thaliana seedlings, co-application of BRs and GA, or BRsand auxin, resulted in a synergistic increase in hypocotyl elongation[66]. An interaction of ABA and BRs in cell elongation has beenshown in Arabidopsis thaliana bee1, bee2, bee3 mutants [19]. Thesemutants were less responsive to exogenous brassinolide (BL) inhypocotyl growth assays, and the triple mutant phenotype wassimilar to bri1 in terms of reduced hypocotyl growth and floralorgan size. Expression of proteins named BR Enhanced Expression(BEE1, BEE2 and BEE3) was repressed by ABA treatment. ABA isa known antagonist of BR signalling. Reduced ABA response inplants overexpressing BEE1 suggests that BEE proteins may func-tion as signalling intermediates in multiple pathways. It is possiblethat BR plays a role in down-regulating some responses to ABA orwhen BRs is lacking may amplify or modify the ABA signal.However, another study suggested that ABA and BR both enhancedrought tolerance in sorghum (Sorghum vulgare) [75].

One of the signal molecules which integrated in the regulationof stress response and plant development is jasmonic acid (JA). Thebiosynthesis of JA is initiated by oxygenation of a-linolenic acid to

Fig. 1. Generalized model of the plant stress signalling networks. ABA, abscisic acid;BRs, brassinosteroids; ET, ethylene; JA, jasmonic acid; SA, salicylic acid.

13-hydroperoxy linolenic acid through the action of a lipoxygenase.On the other hand, 13-hydroperoxy linolenic acid is metabolized byallene oxide synthase (AOS). Subsequently, 12-oxophytodienoicacid (OPDA) is formed by allene oxide cyclase (AOC). After thereduction of a double bond in the cyclopentenone ring of OPDA to12-oxo-phytoenoic acid (OPC8:0) by 12-oxo-phytodienoic acidreductase (OPR). Thus, JA is synthesized from OPDA by the inter-mediates OPC8:0, OPC6:0 and OPC4:0 [57]. The expression of theOPR3 gene is induced by BRs, JA, and by a variety of stimuli like UV-light, touch, wind or wounding. As OPR3 expression is increasedthrough treatment with BRs, it indicates a potential link betweenBR action and JA biosynthesis. BRs may thus influence the stressresponses 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. ROShave also been implicated as important regulatory and signallingelements in a variety of cellular processes. ROS are constantlyproduced during the course of photosynthesis and respiration,whereas redox homeostasis in the cell is tightly controlled byredundant protective mechanisms. Disruption of these protectivemechanisms can cause oxidative stress, leading to oxidativedamage and death of cell. Although, under normal growth condi-tions, the production of ROS in cells is very low, many stresses thatdisrupt the cellular homeostasis of cells enhance the production ofROS. These stresses include drought stress and desiccation, saltstress, chilling, heat shock, heavy metals, ultraviolet radiation,ozone, mechanical stress, nutrient deprivation, pathogen attackand 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 develop-ment, have been implicated in many physiological responses [54].However, little is known about the role of BRs in the plant responseto oxidative stress. It was shown that exogenous application of BRsmodified antioxidant enzymes such as superoxide dismutase,catalase, glutathione peroxidase and ascorbate peroxidase andnon-enzymatic antioxidants, such as ascorbic acid, tocopherols,carotenoids, glutathione, etc. in plants under different stressconditions. Vitamins C, E, and glutathione react directly or viaenzyme catalysis with $OH, H2O2 or O2

$�, while carotenes directlyoperate as effective quenchers of reactive intermediary forms ofoxygen [42,49,50,69].

When maize (Zea mays) seedlings treated with BL were sub-jected to water stress, the activities of superoxide dismutase (SOD),catalase (CAT), ascorbate peroxidase (APX), as well as ascorbic acidand carotenoids contents increased [42]. On the other hand, BRsenhanced the activity of CAT and reduced the activities of peroxi-dase and ascorbic acid oxidase under osmotic stress conditions insorghum (Sorghum vulgare) [69].

Rice seedlings exposed to saline stress and treated with BRshowed a significant increase in the activities of CAT, SOD andglutathione reductase (GR) and a slight increase in APX [49]. Acomparison between salt-sensitive and salt-resistant varieties ofrice treated with 24-epiBL was also reported [50]. 24-epiBL treat-ment, at least in part, improved the tolerance of salt-sensitiveseedlings to short-term salt stress. The difference in alteration ofantioxidant enzyme activities in salt-sensitive rice cultivar maysuggest that the higher salt stress resistance in sensitive seedlingsinduced by 24-epiBL may be due in part to a maintained higheractivity of APX under salt stress conditions.

Algae Chlorella vulgaris respond to heavy metals (cadmium,copper and lead) by induction of several antioxidants, includingseveral enzymatic systems, and the synthesis of low molecular

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weight compounds. Treatment with BL was effective in increasingthe activity of antioxidant enzymes (CAT, GR, and APX) and thecontent of ascorbic acid, carotenoids and glutathione (Bajguz,unpublished data).

The influence of 24-epiBL on some enzymatic antioxidants intomato leaf disc under high (40 �C) temperature was reported [45].BR increased the activity of CAT, peroxidase and SOD in respond tohigh temperature in tomato leaves.

It has been shown that the det2 Arabidopsis mutant, which isblocked in the biosynthetic pathway of BRs [13], had significantlythicker leaves with thickened cuticle layers and cell walls in theepidermal and mesophyll layers, increased stomatal density andcompacted leaf structure with less intercellular space than thewild-type when grown under normal oxygen conditions. Additionof BL to the growth medium resulted in leaves that were moresimilar in morphology to those of Columbia. Interestingly, the det2mutant was also found to be insensitive to the dwarfing effects ofvery low O2 (2.5 kPa) and furthermore did not show the purplingstress response of wild-type of Arabidopsis. BRs could be as possiblemediators of particular aspects of the developmental response tohypoxia, a conclusion supported by the fact that BRs requiremolecular oxygen at several steps in their biosynthesis [51]. It hasbeen also demonstrated that ATPA2 and ATP24a genes encodingperoxidases were constitutively upregulated in the det2 Arabidopsismutant [24]. Furthermore, the oxidative stress-related genesencoding monodehydroascorbate reductase and thioredoxin, thecold and drought stress response genes COR47 and COR78, and theheat 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 stressresistance in det2 plants was correlated with a constitutive increasein SOD activity and increased transcript levels of the defence geneCAT. Therefore, a possible explanation for the fact that the det2mutant exhibited an enhanced oxidative stress resistance is thatthe long-term BR deficiency in the det2 mutant results in a constantin vivo physiological stress that, in turn, activates the constitutiveexpression of some defence genes and, consequently, the activitiesof related enzymes. If this is the case, it may suggest that endoge-nous BRs in wild-type plants somehow act to repress the tran-scription or post-transcription activities of the defence genes toensure the normal growth and development of plants. However, itis still unclear whether BRs directly or indirectly modulate theresponses of plants to oxidative stress [12].

3.3. Response to osmotic stress

Drought, salinity and freeze-induced dehydration constitutedirect osmotic stresses; whereas chilling and hypoxia can indirectlycause osmotic stress via effects on water uptake and loss. Plantsrespond to dehydration and low temperature with a number ofphysiological and developmental changes. Plants have evolveda 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 producehigh levels of abscisic acid (ABA); exogenous application of ABAalso induces a number of genes that respond to dehydration andcold stress. Nevertheless, the role of ABA in low-temperature-responsive gene expression is not clear. Several reports havedescribed genes that are induced by dehydration and lowtemperature. It is likely, therefore, that both ABA-independent andABA-dependent signal transduction cascades exist [67].

Proline is accumulated in many plant species in response toosmotic 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 dehydroge-nase (PDH). These enzymes catalyze the rate-limiting steps of

proline biosynthesis and degradation, respectively. Stimulation ofproline synthesis by ABA and salt stress is correlated with a strikingactivation of P5CS1 expression. By contrast, P5CS2 is only weaklyinduced, whereas PDH is inhibited to different extent by ABA andsalt stress in light-grown Arabidopsis thaliana. However, P5CS2 isalso down-regulated, whereas PDH expression is significantlyenhanced in dark-grown shoots of Arabidopsis. In comparison,P5CS1 expression is inhibited in dark-grown plants. The inhibitoryeffect of dark adaptation on P5CS1 is mimicked by brassinolide (BL).However, BL fails to stimulate PDH, and inhibits P5CS2 only inshoots. Proline accumulation and induction of P5CS1 transcriptionare simultaneously enhanced in the ABA-hypersensitive prl1 andBR-deficient det2 mutants, whereas P5CS2 shows enhancedinduction by ABA and salt only in the det2 mutant. Salt stress isexpected to stimulate an ABA-signalling pathway, which requiresthe ABA1 and ABI1 functions and triggers transcriptional activationof P5CS1 by an ABA-responsive promoter element. The fact, thatboth ABA and salt induction of P5CS1 transcription is inhibited byBL in light-grown plants suggests that BRs may negatively regulatethis common salt and ABA response pathway. Alternatively, BL mayinhibit the light or sugar (or both) regulated maintenance of basalP5CS1 transcription, which is essential for further induction by saltand ABA [1].

The effect of BRs on changes in root nodulation of Phaseolusvulgaris and contents of endogenous ABA and cytokinin trans-zeatin riboside (ZR) has been reported [68]. BRs in the unstressedplants increased root nodulation, ZR content and also amelioratedtheir stress induced decline in the nodulated roots. However, ABAcontents in the nodules of control or stressed plants were notaltered by BRs treatment. Water stress induced decline in rootnodulation is associated with increase in ABA and decline in cyto-kinins contents in the nodulated roots. BRs have the potential toimprove root nodulation and pod yield in the irrigated and waterstressed plants; an effect that could be mediated through aninfluence on cytokinin content in the nodulated roots of Phaseolusvulgaris.

BRs application also resulted in enhancement of seedlinggrowth, which was evident in terms of seedling length, seedlingfresh and dry weights of all the three varieties of sorghum (Sorghumvulgare) viz. CSH-14 and ICSV-745 (susceptible to water stress) andM-35-1 (resistant to water stress) under osmotic stress [69]. Similarresults have been shown in sugar-beet plants under drought stress,in which a reduction of taproot weight was correlated to stressseverity. Treatment with BR fully compensated for the reduction inbiomass caused by mild drought stress. On the other hand,increases in biomass was correlated with increases in acid invertaseactivity in young leaves, which likely provided more assimilates tothe plant due to their larger sizes [58]. Furthermore, osmotic stressresulted in considerable reduction in the protein contents in all thethree varieties of sorghum. However, BRs not only restored but alsostimulated the level of protein and free proline [69].

HomoBL also had a stimulatory effect on the growth of drought-tolerant (C306) and drought-susceptible (HD2329) wheat (Triticumaestivum) varieties under stress conditions. Application of homoBLresulted in increased relative water content, nitrate reductaseactivity, chlorophyll content and photosynthesis under bothconditions. It also improved membrane stability (lower injury).These beneficial effects resulted in higher leaf area, biomassproduction, grain yield and yield related parameters in the treatedplants. However, homoBL showed a higher activity in drought-tolerant wheat variety under water stress conditions. Increasedwater uptake and membrane stability and higher carbon dioxideand nitrogen assimilation rates in BR-treated plants under stresswere correlated with BR-induced drought tolerance [53]. BRs alsoimproved the protoplasmic drought tolerance of suspended cellsfrom hydrated leaves of Sporobolus stapfianus. Hydrated leaves of

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the resurrection grass are not desiccation tolerant, but tolerancecan be induced by drought stress [23].

3.4. Response to heavy metals stress

Plants have a remarkable ability to take up and accumulateheavy metals from their external, for example aquatic environment.Metal contamination of the aquatic environment occurs as a resultof human activities and affects organisms at the biochemical,cellular, community and population level. Aquatic plants areprimary producers of most aquatic food chains and account formuch of the production base of freshwater and marine ecosystems.They have been used to remove heavy metals from aquatic envi-ronments since they have a high capability for accumulating dis-solved metals without dying. High concentrations of all metals,including those essential for growth and metabolism, exert toxiceffects on the metabolic pathways of plants. Toxicity mechanismsinclude the blocking of functional groups of important molecules,e.g. enzymes, polynucleotides, transport systems for essentialnutrients and ions, displacement and/or substitution of essentialions from cellular sites, denaturation and inactivation of enzymes,and disruption of cell and organellar membrane integrity. In addi-tion, metals are also pointed to exert toxic effects through freeradical formation, the phenomenon found to be least explored [63].

Heavy metal toxicity can elicit a variety of adaptive responses inplants. A ubiquitous mechanism for heavy metal detoxification isthe chelation of the metal ion by a ligand. Such ligands includeorganic acids, amino acids, peptides and polypeptides. Peptideligands include the phytochelatins (PC), small gene-encoded,cysteine-rich polypeptides. They are heavy metal-binding peptidesderived from glutathione and have the general structure (g-Glu-Cys)n-Gly, where n has been reported as being as high as 11, but isgenerally in the range of 2–5. PC detoxifies intracellular metals bybinding them through thiolate coordination [14]. BRs stimulatedthe synthesis of PC in Chlorella vulgaris cells treated with lead. Thestimulatory activity of BRs on PC synthesis was arranged in thefollowing order: brassinolide (BL) > 24-epiBL > 28-homo-BL > castasterone (CS) > 24-epiCS > 28-homoCS [9].

The cultures of Chlorella vulgaris treated with BRs and heavymetals show a lower bioaccumulation of heavy metals than thecultures treated with metals alone. The inhibitory effect of BRsmixed with different heavy metals on their accumulation wasarranged in the following order: zinc > cadmium > lead > copper.A stimulatory effect of BRs after blocking the accumulation of heavymetals on the growth and development of Chlorella vulgaris occurs.Application of BRs to Chlorella vulgaris cultures reduced the impactof heavy metals stress on growth, prevented chlorophyll, sugar andprotein loss and increased PC synthesis. Concentration-dependentstimulation was observed with increasing concentration of BR anddecreasing concentration of heavy metals [8,9]. BRs also reducedthe content of cadmium in the seedlings of winter rape [35] andcopper in Indian mustard [61]. BR eliminates the toxic effect ofcadmium on photochemical pathways in rape cotyledons, mainlyby diminishing the damage in reaction centres and O2 evolvingcomplexes as well as maintaining efficient photosynthetic electrontransport [35]. The accumulation of heavy metals (cadmium,copper, lead and zinc) under the influence of BR has been studiedfor different agricultural plants such as barley, tomato, radish andsugar beet. It was found that the application of 24-epiBL signifi-cantly reduced the metal absorption; for example, the content oflead in beet roots was more than 50% lower than in the controlculture [38]. Moreover, Bilkisu et al. [11] reported that brassinolide(BL) during aluminium-related stress stimulated growth in Pha-seolus aureus. It was shown that changes in the ions/metal contentwere influenced by 24-epiBL and dependent on the stage of plantdevelopment 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] reportedthat BRs enhanced activity of the antioxidant enzymes (catalase,peroxidase and superoxidase) and proline content. It has beenfound a significant correlation with the degree of improvement,which was measured in terms of nodulation, nitrogen fixation,pigment composition, carbonic anhydrase and nitrate reductaseactivities. A similar pattern of response together with an elevationin the photosynthesis was observed in the plants of mustardexposed to cadmium through nutrient medium [29]. The foliarspray of either with 24-epiBL or 28-homoBL significantly enhancedthe growth, photosynthesis, antioxidant enzymes and prolinecontent in aluminium stressed mungbean plants [3]. The activitiesof the enzymes catalase, peroxidase, carbonic anhydrase andnitrate reductase also exhibited a significant enhancement inmustard plants grown under nickel stress [2]. These plants alsoexhibited an elevation in the relative water content and photo-synthetic performance. Ali et al. [5] also reported that 24-epiBLenhanced the level of antioxidant system (superoxide dismutase,catalase, peroxidase and glutathione reductase and proline), bothunder stress and stress-free conditions. The influence of 24-epiBLon the antioxidant system was more pronounced under a stresssituation, suggesting that the elevated level of antioxidant system,at least in part, increased the tolerance of mustard plants to salineand/or nickel stress, and thus protected the photosyntheticmachinery and the plant growth.

One of mechanisms for the metal removal is biosorption. Themechanisms of biosorption are generally based on physico-chem-ical interactions between metal ions and the functional groupspresent on the cell surface, such as electrostatic interactions, ionexchange and metal ion chelation or complexation. Functionalgroups most commonly implicated in such interactions includecarboxylate, hydroxyl, amine and phosphoryl groups presentwithin cell wall components such as polysaccharides, lipids andproteins. The binding process is independent of metabolism andhence, of a physical nature. It is also usually rapid and reversible. Inaddition, biosorption can be affected by pH and the presence ofother ions in the medium which may precipitate heavy metals asinsoluble salts, but it is unaffected by metabolic inhibitors or light/dark cycles [71]. Water pH is an important factor directly affectingthe 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 thetoxicity of heavy metals (copper, lead, cadmium or zinc). Lower pHmay increase the bioavailability of metal ions resulting in increasedtoxicity. BR-induced Chlorella vulgaris growth stimulation dependsat least partly on the decrease of pH in the wall space, and thus onacid-induced wall loosening. The effects of BRs on proton secretionare associated with an early hyperpolarization of the trans-membrane electrical potential. BR-induced proton excretion is alsostimulated by the presence of Kþ in the medium [8].

3.5. Response to saline stress

The application of 24-epiBL resulted in substantial improvementin the seed germination and seedling growth of Eucalyptus camal-dulensis under saline stress. Seed germination in the presence of150 mM NaCl was enhanced by 24-epiBL, but when seedlings weregrown hydroponically in salt, uptake of 24-epiBL through rootscaused more damage [55]. BRs removed the salinity-inducedinhibition of seed germination and seedling growth in case of rice(Oryza sativa). BRs also restored the level of chlorophylls andincreased nitrate reductase activity under salt stress. The activity ofthis enzyme plays a pivotal role in the supply of nitrogen and thegrowth and productivity of plants, especially in cereals. The

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reduced nitrate reductase activity in the leaves of salt-stressedplants is attributed to salinity inhibited nitrate transport to theshoot, which in turn is due to interference with nitrate uptake andxylem loading [6]. Likewise, seed soaking of 28-homoBL enhancedthe nitrogen fixing capability in chickpea plants pre-treated withNaCl [4]. The plants also showed increased dry matter accumula-tion, together with an increase in the activities of nitrate reductaseand carbonic anhydrase activities. The 28-homoBL treated plantsalso possessed higher seed yield in comparison to the plants sub-jected to NaCl stress, at harvest. Similarly the spray of 28-homoBLto the foliage or supply through roots of Brassica juncea plantsgenerated from the seeds soaked in NaCl enhanced the growth,nucleic acid content, ethylene and seed yield [28,30,31].

Wheat germ agglutinin (WGA), a classic cereal lectin, level isincreased in plants under unfavourable conditions: fungal infec-tion, drought and osmotic stress, salinity and hyperthermia. ABA isinvolved in the control of WGA synthesis and accumulation. It wasdemonstrated that BRs did not influence on ABA content butenhanced only the accumulation of lectin under salt stress in theroots of wheat (Triticum aestivum). This indicates the possibility ofthe ABA-independent control of the lectin content by BRs. Salttreatment resulted in rapid ABA accumulation, which occurredprior to 5-fold increase in the level of lectin. On the other hand,combined treatment with BRs and NaCl resulted in partial growthrecovery as compared to the action of NaCl alone. BRs reduced thesalinity-induced accumulation of BRs and WGA in roots by 50%. It isprobable that BRs could be involved in the hormonal control of theWGA level along with ABA. BRs evidently exert a protective actionon wheat seedlings via a considerable decrease in the salt-inducedABA and WGA accumulation in roots [59,60].

3.6. Response to thermal stress

The lamina-inclination test is frequently used to evaluate theactivity of brassinosteroids. BRs promoted the cell elongation of riceseedlings at a low temperature (15 �C). On the other hand, the effectof indole-3-acetic acid (IAA) on the cell elongation was markedlylowered, while the combination of BRs and IAA synergisticallypromoted the cell elongation. Treatment with BRs also enhancedthe germination rate of seeds and the growth after direct sowing insubmerged paddy pots at low temperature. Furthermore, leafspraying of BRs on the rice seedlings at the 4th leaf stage increasedplant height and the fresh weights of tops and roots under chillingstress. BR sprayed on the completely expanded 4th leaves did notincrease their blade length, but that sprayed on the expanding 5thand 6th leaves strikingly increased their blade length. These resultsare the evidence that BRs were more effective on the protective ofold rice leaves against cold stress [20,33]. The similar ability of BRsto improve seed germination and seedling growth of maize (Zeamays) [32] and cucumber (Cucumis sativus) [38] under chillingstress has been early reported.

Extreme temperatures (7 and 34 �C) increased stress symptoms,i.e. necrotic areas on the leaves of bananas. However, in plantstreated with a trihydroxylated spirotane analogue of BR the effectsof thermal stress were significantly reduced. Cool temperatureaffected leaf emergence with a significant reduction in theirnumber, but application of BR analogue had marked positive effect.Plant height was also significantly reduced by both temperatureextremes, whereas the application of BR analogue was effectiveonly in plants exposed to the warmer temperature [25].

Application of 24-epiBL minimally increased freezing toleranceof bromegrass (Bromus inermis) cells by 3–5 �C, but markedlyenhanced cell viability following exposure to high (40–45 �C)temperature stress. The net effect on hardening was less than thatobtained with ABA. Treatment of cells with 24-epiBL increased theaccumulation of a subset of ABA-inducible heat-stable proteins.

However, but unlike ABA, 24-epiBL did not induce the expression ofdehydrin transcripts which hybridized to a barley dehydrin clone.Furthermore, 24-epiBL slightly increased the hsp90 transcriptlevels, while ABA reduced the level of expression. These resultsconfirm that 24-epiBL confers some stress tolerance to plant cellsand further suggest that the mechanisms by which thesecompounds exert anti-stress effects may only in part be similar tothat of ABA [73].

Treatment of Brassica napus and tomato seedlings with 24-epiBLleads to an increase in the basic thermotolerance. It resulted inhigher accumulation of four major classes of heat-shock proteins(hsps)dhsp100, hsp90, hsp70, and low-molecular-weight hspsdascompared to untreated seedlings. The higher level of hsps in 24-epiBL-treated seedlings did not correlate with hsp mRNA levelsduring the recovery period. This suggests that 24-epiBL treatmentlimits the loss of some of the components of the translationalapparatus during a prolonged heat stress and increases the level ofexpression of some of the components of the translationalmachinery during recovery. It correlates with higher hsp synthesisduring heat stress, a more rapid resumption of cellular proteinsynthesis following heat stress, and a higher survival rate [16,17].24-epiBL also induced the expression of mitochondrial small hspsin tomato leaves. BR treated tomato plants had better photosyn-thetic efficiency. Significantly higher in vitro pollen germination,enhanced pollen tube growth and low pollen bursting have beenobserved in the presence of 24-epiBL at 35 �C, a temperature highenough to induce heat-stress symptoms in tomato, indicatinga possible role of BR during plant growth and reproduction. Thebeneficial effect of BR application was also observed in fruit yield,which was increased during heat-stressed conditions. This increasein fruit yield was mainly due to increase in fruit number by 24-epiBL application [62].

The effects of high-temperature stress in BR-treated anduntreated wheat leaves were examined at the level of total proteinsynthesis and leaf cell ultrastructure. Protein synthesis was main-tained in BR-treated leaves at 43 �C at levels similar to those at23 �C, whereas in untreated leaves it decreased 2.5-fold at 43 �C ascompared to samples at the control temperature [41]. During heatstress in plants, small hsps aggregate to produce highly orderedcytoplasmic complexes referred to as heat shock granules (HSGs). Itis believed that HSGs represent storage and protection sites forhousekeeping mRNPs, which are released following removal ofstress [41]. It has been also observed that HSGs in wheat leaf cellsaggregated in small clusters. The average number of HSGs in clus-ters and the average cluster size were both higher in BR-treatedleaves than untreated wheat leaves [41].

Kagale et al. [36] showed that although BR can augment toler-ance to heat stress, it is not essential for hsp expression during heatstress treatments in BR-deficient Arabidopsis mutants (det2-1 anddwf4). It suggests that BR deficiency itself is a form of cellular stressor a developmental state that leads to activation of the hsp genes.Since hsps are also expressed in response to developmental andother stress signals. Probably HSP activation in the absence of stressin BR-deficient must occur via other hormones and signallingcomponents.

BR not only significantly promoted epicotyl elongation of mungbean, but also allowed the plant to partially recover from thegrowth-inhibiting effects of chilling. Using proteomics, it has beenidentified some down-regulated proteins that were re-up-regu-lated after BR treatment under chilling conditions. Most of theseproteins were predicted to be involved in cell growth, wallformation, ATP production, the stress response, and methionineassimilation. However, the cross-talk between BR’s action andmethionine’s is minimal. Therefore, the possible involvement ofmethionine assimilation downstream of BR’s action may beconsidered a novel project for future research [34].

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3.7. Response to pathogen stress

Plant immunity is based on a complex response that is highlyflexible in its capacity to recognize and counteract differentinvaders. To combat invasion by microbial pathogens and herbiv-orous insects effectively, plants make use of pre-existing physicaland chemical barriers, as well as inducible defence mechanismsthat become activated upon attack. Apart from reacting locally,plants can mount a systemic response that establishes an enhanceddefensive capacity in tissues distant from the site of primary attack.These include systemic acquired resistance (SAR), which is trig-gered by necrotizing pathogens; induced systemic resistance (ISR),which is activated upon colonization of roots by selected strains ofnon-pathogenic rhizobacteria; and wound induced defence, whichis typically elicited upon tissue damage such as that caused byfeeding insects. Induced defence responses are regulated bya network of interconnecting signal transduction pathways inwhich the hormonal signals salicylic acid (SA), jasmonic acid (JA),and ethylene play a major role, and other hormones such as BRs andABA can also be involved [18].

All known phytohormones are involved in regulation of tuberdormancy. The inhibition of sprouting during deep dormancy ofpotato tubers is connected with the effect of ABA, while thebreakdown of dormancy is accompanied with a decrease in thecontent of ABA and an increase in the content of gibberellins inapical meristems. Potato tubers were shown to produce lessethylene during deep dormancy than on sprouting. The stimulationof endogenous ethylene synthesis may lead to increased synthesisof ABA and prolonged deep dormancy of potato tubers. On the otherhand, BRs stimulated ethylene synthesis in intact potato tubers andABA synthesis in cells of tuber apices. BR-induced changes correlatewith the duration of deep dormancy that also increases. Electronmicroscopic and morphometric studies of functionally differentregions of potato tuber apices showed that the inhibition of cellstretching is one of the BR effects on the cell level. The mostsensitive to BR was the rib meristem, which plays a leading part atthe initial stages of growth of potato tuber apieces [39].

Application of low concentrations of BR-containing (24-epi-castasterone and 24-episecasterone) extract of Lychnis viscaria seeds,yielded an increased resistance of tobacco, cucumber and tomato toviral or fungal pathogens (tobacco mosaic virus, Sphaerotheca fuli-ginea, Botrytis, respectively). However, no direct anti-fungal effectsin mycelium growth assays with Phytophthora infenstans have beenobserved. The activation of the plant’s defence status was correlatedwith a stimulation of three pathogenesis-related (PR) proteins, suchas chitinase, b-1,3-glucanase and peroxidase, which are molecularmarkers of SAR [52]. It has been also reported that BR biosyntheticArabidopsis mutant, cpd, showed a remarkably low expression of PRgenes PR1, PR2, and PR5, with significant induction of them if CPDwere overexpressed in transgenic plants [65].

BRs induced susceptibility of the potato tuber tissues by stim-ulating the mycelial growth and intensity of spore formation ofPhytophthora infenstans and by weakening the immune status ofplant tissues, as evidenced by the inhibition of wound reparation.The immunoinhibiting effect of BRs is a systemic and long-termcharacter. It was observed for at least 4 months after the treatmentof whole potato tubers with BRs [70]. It was also shown thattreatment of potato plants or tubers after harvesting led toa prolongation of the period of deep dormancy of tubers and toenhancement of their resistance to Phytophthora infenstans infec-tion. Under the influence of BRs the ethylene synthesis by tubertissues increased and the biosynthesis of protective substances ofphenolic and terpenoid nature was also activated. It means that theprotective or deprotective type of BRs activity depended onthe method and time of BR application and was connected with thedifferent stimulating points of either the plant or the pathogen [39].

In tobacco, BL enhanced resistance to the viral pathogen tobaccomosaic virus (TMV), the bacterial pathogen Pseudomonas syringaepv. tabaci (Pst), and the fungal pathogen Oidium sp. In rice plants, BLenhances resistance to the fungal pathogen Magnaporthe grisea andthe bacterial pathogen Xanthomonas oryzae pv. oryzae. Measure-ment of SA accumulation and analysis using NahG transgenictobacco revealed that the BL mediated resistance enhancementdoes not require SA in tobacco. During induction of the resistance,BL itself does not induce the expression of the pathogenesis-related(PR) gene in either tobacco or rice. Analysis using brassinazole2001, a specific inhibitor for biosynthesis of BRs, and themeasurement of BRs in TMV-infected tobacco leaves indicate thatsteroid hormone-mediated disease resistance (BDR) plays part indefence response in tobacco. Simultaneous activation of SAR andBDR by SAR inducers and BL, respectively, exhibited additiveprotective effects against TMV and Pst. It indicates that there is nocross-talk between SAR- and BDR-signalling pathway downstreamof BL. Brassinolide enhanced resistance independently of SA andthe combinations of BL and SAR inducers exhibit additive promo-tive effect against pathogens. This means that the SA-mediatedsignalling pathway for induced resistance is distinct from the JA/ethylene- and BL-mediated signalling pathways, however, cross-talks may exist between them [48].

Field application of 24-epiBL (5–15 mg ha�1) to barley plantssignificantly decreased the extent of leaf diseases induced by mixedfungal infection, along with an increase in crop yield. A comparisonof the results with those obtained after application of Bayleton(0.5 kg ha�1), a fungicide, showed the higher capacity of 24-epiBLas a protective factor against fungi. A protective effect of 24-epiBLagainst fungi was also established in field trials with cucumber. Anincrease in the activity of peroxidase and polyphenoloxidaseenzymes in the leaves of cucumber plants has been also found.Since these enzymes are involved in the metabolism of poly-phenols, a change in their activity may be considered as one of thefactors connected with increased plant resistance to infection [39].

Systemin and other primary signals that are released at thewound site promote proteinase inhibitor gene expression by acti-vating the octadecanoid pathway for JA biosynthesis. Systeminbinds to a cell surface receptor (SR160) that was recently identifiedas a leucine-rich repeat receptor kinase. Interestingly, SR160 alsofunctions in tomato as a receptor for BRs. This peptide induces theplant’s defences by increasing the level of proteinase inhibitor andthe expression of polyphenol oxidase genes in both infected andhealthy leaves. Using radiolabelled systemin to purify the protein,SR160 was identified as a leucine-rich repeat receptor-like kinase(LRR-RLK) that shares high homology with Arabidopsis BRASSI-NOSTEROID INSENSITIVE1 (BRI1). The greatest homology betweenSR160 and BRI1 is found in the protein-kinase domain, whereas theextracellular domains vary to a greater extent. The extracellulardomains of both SR160 and BRI1 include a leucine-rich repeat (LRR)region, with an island region between the 21st and 22nd repeat.This island region is thought to play an important role in the ligandbinding of BRI1. There is considerable evidence that BRI1 binds BRsto activate a signalling pathway that controls growth and devel-opment in Arabidopsis, and new data suggest the same for tomato.A novel curl3 (cu3) allele has been identified while screeningtomato plants for BR-signalling mutants. The cu3 mutant had bothincreased levels of endogenous CS and increased expression of theDwarf gene, a transcript normally down-regulated by BR. Degen-erate primers amplified a product that corresponded to BRI1, andproducts from cu3 mutant plants contained nonsense mutations inthe kinase domain, showing that CU3 encodes a BRI1 orthologue.The putative tomato BRI1 gene (tBRI1) has only polymorphicdifferences when compared to SR160, the recently identified sys-temin receptor. This suggests the possibility that a single receptor-like kinase (RLK), SR160/tBRI1, can bind systemin and BRs. This is

A. Bajguz, S. Hayat / Plant Physiology and Biochemistry 47 (2009) 1–8 7

the first indication that one RLK can respond to multiple types ofligands and be involved in several pathways that have significantlydifferent functions [64,72].

3.8. Response to pesticides application

Pesticides may be a chemical substance or biological agent (suchas a virus or bacteria) used against pests including insects, plantpathogens, weeds, molluscs, birds, mammals, fish, nematodes andmicrobes that compete with humans for food, destroy property,spread disease or are a nuisance. Modern pesticides are designed toensure the effectiveness against target organisms. Many pesticideshave been developed to target specific biochemical reactionswithin their target organism. However, it is of great concern tounderstand their effects on non-targeted crops they are meant toprotect [43].

BRs are reported to be effective in reducing damage caused bypesticides. Effects on herbicide safening, where BL treatmentreduced the damage from treatment of rice with simazine, buta-chlor, or pretilachlor have been confirmed [54]. The phytotoxiceffect on cucumber leaves of nine pesticides, including threeherbicides (paraquat, fluazifop-p-butyl, and haloxyfop), threefungicides (flusilazole, cuproxat, and cyazofamid) and three insec-ticides (imidacloprid, chlorpyrifos, and abamectin) has beenexamined [74]. Plants treated with paraquat showed the severestphytotoxic symptom with the highest reduction in net photosyn-thetic rate (Pn), while other pesticides except flusilazole inhibitedPn to various degrees. The inhibition of Pn by cuproxat was

Fig. 2. Effects of brassinosteroids on plants exposed or subjected to various stresses.

accompanied by declines both in stomatal conductance and inter-cellular CO2 concentration, whereas decreased Pn for the cyazofa-mid was associated with increased intercellular CO2 concentration.For other pesticides, inhibition of Pn was accompanied by decreasein stomatal conductance, while intercellular CO2 concentration wasincreased or unaffected. Interestingly, inhibitions of Pn were alle-viated by 24-epiBL pre-treatment, as for the pesticides exceptparaquat and flusilazole. Pesticides application impairs the photo-synthesis of cucumber seedlings, while 24-epiBL pre-treatment canincrease the resistance of plants to pesticides, which might bemediated by enhanced activities of CO2 assimilation.

4. Conclusion

BRs can act efficiently in plants as immunomodulators whenapplied at the appropriate concentration and at the correct stage ofplant development. BRs are implicated in plant responses to abioticenvironmental stresses and to undergo profound changes in plantsinteracting with bacterial, fungal and viral pathogens (Fig. 2). BR-regulated stress response as a result of a complex sequence ofbiochemical reactions such as activation or suppression of keyenzymatic reactions, induction of protein synthesis, and theproduction of various chemical defence compounds. BRs open upnew approaches for plant resistance against hazardous environ-mental conditions.

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