Physiological and molecular effects of 24-epibrassinolide, a brassinosteroid on

thermotolerance of tomato

Ishwar Singh1,2 and Mariko Shono1,*1Japan International Research Center for Agricultural Sciences, Okinawa Subtropical Station, Maezato1091-1, Ishigaki, Okinawa 907-0002, Japan; 2Present Address: Indian Institute of Sugarcane Research, P.O.Dilkusha, Lucknow 226 002, India; *Author for correspondence (e-mail: mshono@jircas.affrc.go.jp; phone:+81-980-88-6202; fax: +81-980-88-6204)

Received 06 October 2005; accepted in revised form 20 September 2005

Key words: Brassinosteroids, Heat-stress, Mitochondrial sHSP, Photosynthetic rate, Pollen germination

Abstract

Brassinosteroids are naturally occurring plant growth regulators, which exhibit structural similarities toanimal steroid hormones. Recent studies have indicated that besides an essential role in plant growth anddevelopment, brassinosteroids also exert anti-stress effects on plants. We show here that tomato plantstreated with 24-epibrassinolide (EBR) are more tolerant to high temperature than untreated plants. Ananalysis of mitochondrial small heat shock proteins (MT-sHSP) in tomato leaves by western blottingrevealed that the MT-sHSP did not preferentially accumulate in EBR treated plants at 25 �C. However,treatment of plants at 38 �C induced much more accumulation of MT-sHSP in EBR treated than inuntreated plants. Results of this study provide the first direct evidence for EBR induced expression of MT-sHSP, which possibly induced thermotolerance in tomato plants. EBR treated tomato plants had betterphotosynthetic efficiency. We also observed significantly higher in vitro pollen germination, enhanced pollentube growth and low pollen bursting in the presence of EBR at 35 �C, a temperature high enough to induceheat-stress symptoms in tomato, indicating a possible role of EBR during plant reproduction.

Abbreviations: A – Net photosynthetic rate; BRs – Brassinosteroids; Ci – Internal CO2 concentration;E – Transpiration rate; EBR – 24-epibrassinolide; gs – Stomatal conductance; MT-sHSP – Mitochondrialsmall heat-shock protein

Introduction

Brassinosteroids (BRs) are natural plant growthpromoting substances, which occur at low con-centrations in all plant parts. Recent studies haveindicated that BRs are essential for proper plantgrowth and development (Arteca 1995; Yokota1997; Sasse 1997; Clouse and Sasse 1998). Sincethe discovery of BRs as a plant growth promotingsubstance in rape pollen (Grove et al. 1979), theoccurrence of BRs has been demonstrated in

almost every plant organ such as pollen, flower,shoot, leaf, fruit and seed (Kim 1991; Fujioka1999). Various studies conducted so far on thephysiological roles of BRs in plants have suggestedthat BRs might be involved in the regulation of cellelongation and division, leaf bending, reproductiveand vascular development, membrane polarizationand proton pump activity, source/sink and mod-ulation of stress (Yokota and Takahashi 1986;Mandava 1988; Sakurai and Fujioka 1993; Arteca1995; Yokota 1997; Clouse and Sasse 1998; Sasse

Plant Growth Regulation (2005) 47:111–119 � Springer 2005

DOI 10.1007/s10725-005-3252-0

1999). The studies using BR- deficient Arabidopsis,pea and tomato revealed that BR-deficiency re-duced stem elongation and fertility (Clouse andSasse 1998; Clouse and Feldmann 1999).

Hamada (1986) reported for the first time theameliorative effects of BR-treatment of stressedplants and since then most of the work has beenfocussed on chilling stress (Katsumi 1991; Wilenet al. 1995). The enhanced chilling resistance wasattributed to BR-induced effects on membranestability and osmoregulation (Wang and Zeng1993). Changes in the spectrum of heat shockproteins after BR administration and promotionof heat shock granule formation had also beenreported in heat stressed wheat (Kulaeva et al.1991). Treatment with 24-epibrassinolide (EBR),enhanced growth of juvenile gram (Cicer arieti-num) plants, increased in situ relative water con-tent and decreased stomatal transpiration rate(Xu et al. 1994) under drought stress and im-proved tolerance against salt in rice (Anuradhaand Rao 2001). The sequential studies on thebrassinosteroid distribution in maturing pollenshowed that the level of free BRs increased as thepollen developed (Asakawa et al. 1996). Dha-ubhadel et al. (1999) reported that BRs inducedthermotolerance in Brassica napus and tomatoseedlings grown on culture media, which wasattributed to BRs induced expression of heatshock proteins (HSPs). The increased accumula-tion of HSPs resulted from higher HSPs synthesis(Dhaubhadel et al. 2002). Recently Singh andShono (2003) provided first ever evidence for 24-epibrassinolide induced thermotolerance in ger-minating pollen. We are interested in the amelio-rative effects of brassinosteroids on plant growthand reproduction during heat-stress and tounderstand the mechanism of EBR induced ther-motolerance, we examined the expression ofmitochondrial small heat shock proteins (MT-sHSPs), which are reported to be involved inthermotolerance of plants (Liu and Shono 1999;Sanmiya et al. 2004).

Materials and methods

About 24-epibrassinolide (EBR) was purchasedfrom Sigma Chemicals, USA. 10 mM stock solu-tion of EBR was prepared by dissolving in abso-lute ethanol. The stock was stored in a deep freeze

at �20 �C and different concentrations wereprepared from this stock as and when required.

Plant material

Tomato (Lycopersicon esculentum Mill.) cultivar‘Ailsa craig’ was grown under greenhouse condi-tions (25 �C day/20 �C night temperatures undernatural day light for 12 h) at JIRCAS, Okinawa,Japan during the year 2000–2001. The seedlingswere raised in small plastic pots (9 cm top diam-eter) filled with 200 g of sand, vermiculite andFYM (2:1:1). Two seedlings per pot were trans-planted while on establishment thinned to one.Three weeks old healthy seedlings were trans-planted to large plastic pots (16 cm top diameter).The plants were irrigated daily and 2 g of fertilizer(NPK, 14:14:14) was applied to each pot afterestablishment of seedlings at fortnightly interval.Desired concentrations of EBR (1, 10 and 20 lM)along with a control (0.01% ethanol) were pre-pared just before use and 12.5 ml solution wassprayed on four weeks old plants with a handspray. Prior to spray the solution (EBR or Con-trol) was mixed with 0.1% Teepol (Teepol acts assurfactant and facilitates absorption of growthregulator by the leaf surface). The pots were ar-ranged in a completely randomized design (CRD)to conduct five independent experiments.

Experiment-I: plant survival at lethal temperature

One week after EBR application, EBR treatedalong with untreated (control) tomato plants wereexposed to 45 �C for 2 and 3 h under a light inten-sity of 200 lE m�2 s�1 and then returned togreenhouse (25/20 �C day–night temperatures) torecover. Seven days later, the plants were scored asdead, alive or recovering. The plants that showedwilting symptoms after heat-shock treatment, butlater recovered at normal temperature were scoredas recovering ones.However, a plant scored as alive,survived heat-shock treatment without wilting.

Experiment-II: expression of MT-sHSP

To study the expression of MT-sHSP, the EBR(1 lM) treated and untreated (control) tomato

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plants in triplicate (10 days after EBR application)were transferred to a growth chamber (G1) main-tained at 25 �C under 12/12 h light–dark cycle for24 h and then transferred to another growthchamber (G2) kept ready at 38 �C. EBR concen-tration was chosen on the basis of the results ofExperiment-I, where most of the EBR (1 lM)treated tomato plants survived on exposure to45 �C for 3 h, which otherwise killed all the un-treated plants. Leaf samples for protein analysiswere collected at 0, 0.5, 1 and 2 h of high tem-perature (38 �C) treatment. After completion ofthe temperature treatment, the plants were againtransferred from growth chamber G2 to G1 tostudy the recovery pattern and samples for proteinanalysis were collected after 24 and 48 h ofrecovery.

Composite sample of leaf tissues (500 mg) wasmacerated separately in 3-ml lysis buffer consistingof 0.25 M sucrose, 20 mM Tris–HCl (pH 7.5),0.5 mM ethylene diamine tetra acetic acid (EDTA)and 2.0% w/v polyvinyl pyrrolidine (PVP) in anice cooled pestle mortar. The homogenate wascentrifuged at 1000 · g for 10 min at 4 �C. Thepellet was discarded and the supernatant was againcentrifuged at 10,000 · g for 30 min at 4 �C. Thepellet was dissolved in 50 ll extraction buffer(20 mM Tris–HCl, pH 7.5 + 0.5 mM EDTA).The samples were prepared by mixing 50 ll sampleextract with 50 ll sample buffer (consisting of60 mM Tris–HCl, pH 6.8, 10% glycerol, 2% SDSand 0.002% BPB). Just before use the samplebuffer was mixed with 2-mercaptoethanol in a ra-tio of 9:1.

For western blot analysis, proteins (5 ll of eachsample) were separated on SDS-PAGE accordingto the method of Laemmli (1970) and transferredon to PVDF membrane by electroblotting usingthe wet type electroblotting cell (Model NA-1510,Nippon Eido, Japan). The MT-sHSPs weredetected by sequential incubation with anti-MT-sHSPs antibodies and peroxidase conjugated anti-rabbit IgG antiserum, each at a dilution of 1:1000,followed by chromogenic visualization by a per-oxidase Kit (Vector Laboratories Inc., Burlingame,California, USA). The anti-MT-sHSP antibodywas produced as per the procedure described bySanmiya et al. (2004). The full-length cDNA forthe tomato MT-sHSP gene was sub-cloned into theglutathione S-tranferase (GST) vector (Amer-sham), an expression vector in E-coli. The GST-

MT-sHSP fusion protein was purified according toinstruction manual and used as the antigen. Rabbitanti-MT-sHSP antibody was produced and affin-ity-purified by Sawady technology. The antibodydid not react with any proteins of 23.8 kDa in anti-sense lines of tobacco for the 23.8 kDa MT-sHSP,thus indicating its specificity to MT-sHSP. Theexperiment was repeated three times and the pat-terns of MT-sHSP accumulation were found to beconsistently reproducible.

Experiment-III: CO2 gas exchange studies

The net photosynthetic rate (A), stomatal con-ductance (gs), transpiration rate (E) and internalCO2 concentration (Ci) were recorded at 2 weeksafter EBR application, with an infrared gas ana-lyzer (Model LCA-4, ADC Hertz, UK) equippedwith air supply unit and Parkinson broad leafchamber (aperture area 6.25 cm2). Measurementswere made within one minute after enclosing theyoungest uppermost fully expanded leaf into thechamber.

After 2 weeks of EBR application, the EBRtreated (1 lM) and untreated (control) tomatoplants (5 plants in each case) were shifted to agrowth chamber (G1) maintained at 25/20 �Cunder a 14/10 day–night cycle for 24 h for accli-matization and then transferred to a growthchamber (G2) kept ready at 35/27 �C day–nighttemperatures. Both growth chambers were main-tained at 600 lmol m�2 s�1 light irradiance and360 ppm CO2. The relative humidity of the twochambers was kept controlled at 65 and 77% inG1 and G2, respectively, to keep the vapor pres-sure deficit at a similar level (1.5 kPa). Measure-ments for An, E, gs and Ci were recorded at 0, 2, 4and 24 h after starting of temperature treatment.Both EBR treated and untreated plants were thentransferred to G1 from G2 after 24 h of temper-ature treatment to study the recovery pattern.Data on different CO2 exchange parameters(A, E, gs, Ci) on these plants was recorded after24 h of recovery.

Experiment-IV: in vitro pollen germination

For this experiment we used untreated plantsgrown under normal greenhouse conditions

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(25/20 �C day–night temperatures with naturalirradiance. Pollen grains were collected on butterpaper between 0900 and 1100 h from freshlyopened flowers. The pollen were mixed thor-oughly and equal quantities were inoculated ongerminating medium described by Singh andShono (2003) that contained 15% sucrose,250 mg l�1 boric acid, 200 mg l�1 calcium nitrateand 0.8% agar. The germinating mixture wasboiled for 5 min in a microwave oven andallowed to cool at a temperature untill the med-ium started solidifying. Wherever EBR at finalconcentrations of 1, 10 and 50 lM was addedquickly to the basal medium, 5 ml was poured ineach petri-plate (6 cm diam) and they wereallowed to cool at room temperature (25±2 �C).These were then inoculated with pollen andplaced in incubators maintained at 25, 35 and40 �C. After 4 h of incubation, the petriplateswere taken out and in each plate 2 ml staining(safranin 0.02 % w/v), killing and fixing solution(glycerol, formaldehyde, glacial acetic acid anddistilled water, 20:5:3:72) was poured. The petri-plates were examined under phase contrastmicroscope (100·). Ten readings per plate forpollen germination and bursting and 20 readingsper plate for pollen tube length were taken byrecording on microscopic field basis.

Experiment V: yield and its attributes

In this experiment 30 days old plants (two sets of10 plants each), maintained at normal greenhouseconditions (25/20 �C day–night temperatures)were sprayed with 12.5 ml of 1 lM EBR or 0.01%ethanol (control). The concentration of EBR(1 lM) was selected on the basis of higher pollengermination under in vitro conditions. This treat-ment was repeated again after 30 days (60 daysold plants) keeping all conditions similar exceptthat the quantity of solution sprayed was 25 mlper plant. One set of pots (having 5 treated and 5untreated plants) was then moved to anothergreenhouse kept ready at 35/27 �C day–nighttemperatures (Heat-stress) and maintained theretill the final harvest. Fruits were harvested twice aweek from both greenhouses and data wererecorded for fruit weight and fruit number. Afterthe final harvest at about 4 months old plants, thedata were pooled to obtain fruit weight and

number per plant and averaged to get meanvalues. The data was analyzed statistically anddifferences among EBR treatment and controlwere evaluated by the least significant difference(LSD0.05).

Results

A lethal temperature for survival of tomato plantswas identified by exposing 1-month-old tomatoplants to different temperatures for varying lengthsof time, and then to 25 �C for recovery. A 3 hexposure to 45 �C reproducibly killed 90–100% ofthe untreated plants. This treatment was thereforeselected to study the effect of EBR treatment ontomato plants subjected to high temperaturestress. EBR treated and untreated tomato plantswere also subjected to 45 �C for 4 h, which killedall the untreated and most of the EBR treatedplants (data not shown). Initial plant growth wasbetter in all EBR treated plants at 25 �C as com-pared to untreated one. Although 2 h exposure to45 �C was insufficient to kill both the untreatedand EBR treated plants, however growth depres-sions were observed in untreated and 20 lM EBRtreated plants. Exposure to 45 �C for 3 h com-pletely killed >90% untreated plants, while 1 lMEBR application was found to be most effectivefor survival of tomato plants at lethal temperature(Figure 1).

The MT-sHSPs were not preferentiallyexpressed at 25 �C but started accumulatingwithin 30 min of a heat shock treatment (38 �C)in leaves of both treated and untreated plants,with not so much difference in level of expression.However, after 1 and 2 h of temperature treat-ment and also during a 24 h recovery, these pro-teins accumulated to a much higher level in EBRtreated plants than in the untreated one (Fig-ure 2), indicating a possible role of EBR in heattolerance of plants.

The A, E and gs were higher in EBR treatedtomato plants as compared to untreated one, whilethere was no significant difference in Ci at 25 �C.Heat-stress decreased the photosynthetic CO2

assimilation rate, however, EBR treated plantsmaintained higher level of A rate as compared tountreated ones (Figure 3a). The E and gs increasedsharply in EBR treated plants during heat-stressFigure 3b, c). The Ci increased sharply in

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untreated plants after 4 h of heat-stress treatmentFigure 3d), indicating their less adaptability tohigh temperature which was reflected in decreasedlevel of net photosynthetic rate.

In vitro pollen germination at high temperatureshowed a varied response to different EBR con-centrations. About 50 lM EBR totally inhibitedpollen germination; however, we observed a sig-nificant increase in in vitro pollen germination with1lM EBR compared with the control at hightemperature (Table 1). Other effects of 1 lM EBRon pollen viability included enhanced pollen tubegrowth and reduced pollen bursting during heatstress.

The fruit yield was significantly increased byapplication of 1 lM EBR both under normal (25/20 �C) as well as heat-stressed (35/27 �C) condi-tions. owever, this ncrease in fruit yield was muchhigher during heat-stress Figure 4). EBR appli-cation also increased the fruit number per lantunder normal as well as heat stress conditions,however, here was no improvement in mean fruitweight by EBR application under normal tem-perature conditions and during eat-stress (datanot shown).

Figure 2. Accumulation of MT-sHSP in tomato leaves during heat stress and recovery. The EBR treated (EBR) and untreated

(control) plants that were exposed to 38 �C for 2 h were allowed to recover at 25 �C for 24 and 48 h (R-24 h, R-48 h). Proteins were

separated by SDS-PAGE and transferred on to PVDF membrane by electroblotting. MT-sHSP were detected by sequential incubation

of the blot with anti-MT-sHSP antibodies and peroxidase conjugated anti-rabbit IgG antiserum followed by chromogenic visualization

with a peroxidase kit Vector Laboratories Inc. California, USA).

Figure 1. Effect of EBR on survival of tomato plants after a

lethal heat treatment. Both treated (EBR) and untreated plants

were exposed to 45 �C for 3 h. After 7 days of recovery at 25/

20 �C, the plants were scored as alive, dead or recovering. The

results represent mean±SEM of three independent experi-

ments. ‘n’ indicates total number of plants used in each exper-

iment. The results in each treatment are distributed in three

outcome categories (alive, dead and recovering).

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Table 1. Effect of EBR on in vitro pollen germination (%), bursting (%) and tube length (lm) in tomato cv. Ailsa craig.

EBR Conc.

(lM)

Incubation temperature (�C)

25 35 40

Pollen Germination (%)

0 91.9±1.3 73.5±1.4 3.5±0.8

1 94.8±1.6 83.7±3.5 4.6±0.9

10 83.2±2.6 41.2±3.3 0.0±0.0

50 0.0±0.0 0.0±0.0 0.0±0.0

Pollen Bursting (%)

0 6.7±2.7 17.8±2.4 82.56±2.1

1 4.3±1.5 13.1±1.3 75.26±3.3

10 11.1±1.2 25.7±4.6 95.00±2.3

50 6.4±2.0 15.1±1.1 28.43±5.1

Pollen Tube Length (lm)

0 1215.8±111.6 493.7±46.0 40.8±9.3

1 1446.4±52.2 534.5±31.1 61.2±7.2

10 1064.9±54.3 42.4±9.7 00.0±0.0

50 0.0±0.0 0.0±0.0 0.0±0.0

Figure 3. Effect of EBR on net photosynthetic rate (A), transpiration rate (E), stomatal conductance (gs) and internal CO2 concen-

tration (Ci) of tomato cv. ¢Ailsa craig¢ under high temperature conditions. Each value represents mean±SD of five replicates.

116

Discussion

Brassinosteroids have been reported to exert anti-stress effects on plants (Mandava 1988; Sasse 1997;Clouse and Sasse 998). However, only limitedstudies have been conducted so far on the role ofbrassinosteroids in thermotolerance of plants(Kulaeva et al. 1991; Wilen et al. 1995; Dhaubh-adel et al. 1999; Dhaubhadel et al. 2002). Most ofthe living organisms possess acquired thermotol-erance (Lindquist and Craig 1988) and they cansurvive at an otherwise lethal temperature, if theyare given a mild heat treatment prior to lethalheat-stress. However, the EBR treatment induceda basic thermotolerance in tomato and Brassicanapus seedlings (Dhaubhadel et al. 1999) and ingerminating tomato pollen (Singh and Shono,2003). We also observed that EBR (1 lM) treatedtomato plant survived at lethal temperature(Figure 1) which could be due to over expressionof MT-sHSP in EBR treated plants (Figure 2).Accumulation of HSPs has been correlated withincreased themotolerance (Parsell and Lindquist1993) and EBR treatment induced expression offour major classes of HSPs, hsp100, hsp90,hsp70 and sHSPs (Dhaubhadel et al. 1999).Accumulation of MT-sHSPs in mitochondriaduring heat-stress has been reported in a numberof plant species. Since heat-stressed plants accu-mulate much more MT-sHSPs (Vierling 1991;Lund et al. 1998), it is suggested that they arepossibly involved in heat tolerance of crop plantsand play a pivotal role in enhancing thermotoler-

ance (Sanmiya et al. 2004). Increased accumula-tion of HSPs in EBR treated plants possiblyresulted from higher HSP synthesis. EBR treat-ment limited the loss of some of the components ofthe translational apparatus during prolonged heat-stress and increased the level of expression of someof the components of the translational machineryduring recovery, which was correlated with a morerapid resumption of cellular protein synthesis fol-lowing heat-stress and a higher survival rate(Dhaubhadel et al. 2002).

EBR treated plants also showed an enhanced A,E and gs (Figure 3), indicating better adaptation oftreated plants to high temperature. Yu et al. (2004)also reported significant increase in net photo-synthetic rate by EBR application in Cucumissativus. The number of unsaturated lipids (fattyacids with double bond) in thylakoid membrane ofchloroplasts- that contain the light absorbing sys-tem, electron transport chain and ATP synthase- isimportant in determining a plant’s ability forgrowth and photosynthesis at high temperature(Murakami et al. 2000). Loss in thylakoid mem-brane thermostability possibly results in reductionof photosynthesis (Bukhov et al. 1999) in un-treated plants. EBR application possibly increasedthe capacity of CO2 assimilation in the calvincycle, which was mainly attributed to an increasein the activity of Rubisco (Yu et al. 2004). Theenhanced E and gs at high temperature possiblyresults in a reduction of leaf temperature (Jin andShen 1999), which might have helped the plant toperform metabolic activities during heat-stress.

Figure 4. Effect of EBR on fruit weight and numer in tomato cv. ¢Ailsa craig¢ under normal and high temperature conditions. Each

value is the mean±SEM of five replicates.

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Pollen germination and tube growth are highlysensitive to heat-stress (Weaver and Timm 1989;Song et al. 1999) and lead to reduction in yield dueto failure of pollination and fertilization. Brassi-nosteroids are reported to enhance pollen tubegrowth in Prunus avium (Hewitt et al. 1985) andtomato (Singh and Shono 2003). We observedsignificantly higher pollen germination with 1 lMEBR both at normal and high temperature(Table 1). EBR treatment markedly reduced pol-len bursting and that could be the possible reasonfor the effect of EBR on pollen fertility. Floweringand fruit set are highly heat sensitive processes oftomato plant (Weaver and Timm 1989; Dane et al.1991) and relate directly to yield. The beneficialeffect of EBR application was also observed infruit yield, which increased by 31 and 160% duringnormal and heat-stressed conditions respectively(Figure 4). This increase in fruit yield was mainlydue to increase in fruit number by EBR applica-tion. To our knowledge this is the first report thatbrassinosteroids can counteract the inhibitoryeffect of high temperature on plant growth andreproduction.

Acknowledgements

The senior author would like to express his sincerethanks to the Director-General, JIRCAS forallowing him to continue this work at OkinawaSubtropical Station under the ‘CounterpartInvitation Fellowship Program’. This research wassupported in part by funds from the Bio-orientedTechnology Research Advancement Institution,Japan.

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