ORIGINAL PAPER

Physiological response to heat stress of tomato ‘Micro-Tom’plants expressing high and low levels of mitochondrial sHSP23.6protein

Cristina Moll Huther • Aline Ramm •

Cesar Valmor Rombaldi • Marcos Antonio Bacarin

Received: 22 October 2012 / Accepted: 31 January 2013 / Published online: 10 February 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Tomato ‘Micro-Tom’ plants were transformed

for high or low expression of the mitochondrial small ‘‘heat

shock’’ protein (HSP) (MT-sHSP23.6) to evaluate their

response to high temperature. The plants were raised for

59 days under a controlled temperature, photoperiod and

photon flow density and then subjected to heat stress for

24 h at 37 �C, followed by a recovery period under normal

conditions (21 ± 2 �C). The cycle was repeated. The

chlorophyll a fluorescence intensity was measured, and

the parameters of the JIP-test were calculated. The gas

exchange was also evaluated. The JIP-test showed signifi-

cantly different responses of the genotypes to heat stress.

The parameters of photosystem I activity and the net

assimilation of CO2 increased during the first stress cycle

in genotypes with a high expression of MT-sHSP23.6 and

in non-transformed plants; however, the net assimilation of

CO2 decreased in genotypes with a low expression of MT-

sHSP23.6. The data suggest that MT-sHSP23.6 participates

in the heat tolerance mechanism, considering that the

suppression of this protein resulted in greater physiological

damage during heat stress.

Keywords Abiotic stress � Chlorophyll fluorescence �HSP � JIP-test � Mitochondria � Photosynthesis

Abbreviations

ABS/RC Absorption flux (of antenna Chls) per RC

AS Plants transformed in the anti-sense orien-

tation

DI0/RC Dissipation flux per RC

ET0/RC Electron transport flux (further than QA-) per

RC

MT-sHSP Mitochondrial small HSP

N Turnover number as reduction, oxidation,

re-reduction of QA in time span from light

until reaching FM

OEC Oxygen evolution complex

PIABS Performance index (potential) for energy

conservation from exciton to the reduction of

intersystem electron acceptors

PItotal Performance index (potential) for energy

conservation from exciton to the reduction of

PSI end acceptors

PSII Photosystem II

PSI Photosystem I

RC Reaction centre

RE0/RC Electron flux reducing end electron acceptors

at the PSI acceptor side, per RC

sHSPs Small HSPs

S Plants transformed in the sense orientation

Sm Total complementary area normalised above

the transient OJIP

Ss Total complementary area normalised above

the curve of only transient OJ

TR0/RC Trapped energy flux (leading to QA reduction)

per RC

WT Non-transformed plants

uPo Maximum quantum yield for primary photo-

chemistry

uEo Quantum yield for electron transport (ET)

C. M. Huther � A. Ramm � M. A. Bacarin (&)

Departamento de Botanica, Instituto de Biologia,

Universidade Federal de Pelotas, Campus Capao do Leao,

Pelotas, RS 96010-900, Brazil

e-mail: marcos.baccarin@gmail.com; bacarin@ufpel.edu.br

C. V. Rombaldi

Departamento de Ciencia e Tecnologia Agroindustrial,

Faculdade de Agronomia Eliseo Maciel,

Universidade Federal de Pelotas, Campus Capao do Leao,

Pelotas, RS 96010-900, Brazil

123

Plant Growth Regul (2013) 70:175–185

DOI 10.1007/s10725-013-9790-y

uRo Quantum yield for reduction of end electron

acceptors at the PSI acceptor side (RE)

uDo Quantum yield for dissipation (DI)

W0 Probability (at time 0) that a trapped exciton

moves an electron into the electron transport

chain beyond QA-

d0 Probability with which an electron from the

intersystem electron carriers moves to reduce

end electron acceptors at the PSI acceptor side

Introduction

Among abiotic stresses that affect plant growth and

development, heat stress induces metabolic alterations and

disturbances that can lead to the shortening of a plant’s life

span. The reversibility of the stress response depends upon

the stress level or adaptation process. Among the physio-

logical processes, photosynthesis is one of the most sen-

sitive to heat stress and can even be inhibited if plants are

subjected to temperatures much higher than the optimum

for their growth. In the photosynthetic machinery, the main

effect is reported to be in photosystem II (PSII) (Oukar-

roum et al. 2012); however, there are reports that other

components, such as the inactivation of reaction centres of

PSII (Bukhov et al. 1990) and electron transfer between QA

and QB (Ducruet and Lemoine 1985), can also be affected

by high temperatures.

Changes in temperature, light, water or hormonal bal-

ance can lead to modifications of gene expression. At the

molecular level, the quantity of ‘‘heat shock’’ protein

(HSP) can increase at high temperatures. When plants are

subjected to five degrees above the optimum temperature,

the synthesis of mRNAs and proteins is strongly affected,

while the transcription and translation of a small set of

HSPs is initiated (Vierling 1991). Several chloroplastic

HSPs of low molecular mass (sHSP) located in the thyla-

koid membrane, associated with the oxygen evolution

complex (OEC), protect but do not repair the damage

caused to PSII during heat stress (Preczewski et al. 2000).

There are also reports of a complex network of defence to

tolerate certain heat-induced stresses, with HSPs as a

response (Baniwal et al. 2004). The HSPs, and especially

the small HSPs (sHSPs), respond to abiotic stresses but can

also act as a tolerance mechanism (Pegoraro et al. 2011).

There is a wide range of studies showing the relation-

ships between environmental conditions and HSP genes,

proteins and physiological responses (Lee et al. 2000;

Murakami et al. 2004). However, in each case, it is nec-

essary to demonstrate if the HSP induction is the cause or

the consequence of the biotic or abiotic stress (Nishizawa

et al. 2006). For example, some works demonstrated that

MT-sHSPs are involved in tobacco (Sanmiya et al. 2004)

and tomato (Nautiyal et al. 2005; Nautiyal and Shono

2010) thermotolerance, although studies are needed to

clarify the functional specificities or similarities of each

MT-sHSP supposedly involved in plant defence. For

example, previous works showed that 23.6 MT-sHSP

transcripts are strongly accumulated (26.5- to 397.5-fold)

in Arabidopsis cultivated under abiotic stress (Charng et al.

2007; Gandia-Herrero et al. 2008), but no experiments

were performed to study its role in tomato plants. Because

23.6 MT-sHSP has a single sequence in tomato, the tomato

could be used to study the specific effects on photosyn-

thetic thermotolerance.

Because mitochondria can regulate the cell response to

stress, the study of the differential expression of mito-

chondrial genes in response to stress can elucidate the

processes involved in the adaptation to different stresses.

Van Akenet et al. (2009) reported that in Arabidopsis

thaliana, the changes in the stress response proteins are not

related to changes at the transcriptional level, thus sug-

gesting that the post-transcriptional mechanisms may play

important roles in defining the mitochondrial response.

Although photosynthesis is a plastid process, the inter-

connection between organelles is well known. Bethune

et al. (2006) proposed mixtures of retrograde and antero-

grade and hypothesised that the maintenance of the

homeostasis of biochemical and physiological events of

plastids is highly dependent upon the mitochondria and

vice versa. Thus, it is hypothesised that any effect on one

organelle can affect the metabolism of other cellular

compartments.

Considering that photosynthesis is the main process

that is affected by heat stress, it is desirable to study the

response of different genotypes. Several techniques are

used to evaluate the photosynthetic process, and the kinetic

analysis of chlorophyll (Chl) a fluorescence is widely used

to study the photosynthetic apparatus, the external factors

affecting photosynthesis, and also the yield of photosyn-

thetic organisms. The changes in Chl a fluorescence are

indicative of alterations in photosynthetic activities. The

efficiency of electron transport through PSII and its oper-

ational efficiency are correlated to CO2 assimilation and

provide a good tool to examine the photosynthetic and

physiological performance of plants (Srivastava et al. 1999;

Baker 2008; Schansker et al. 2011).

The Chl a fluorescence transient shows a polyphase

shape, with the basic steps O-J-I–P, but an additional step

‘‘K’’ appears in the kinetic curve of Chl a fluorescence

OJIP when leaves are exposed to high temperatures

(Strasser 1997; Oukarroum et al. 2012). This step appears

at 300 ls and is most likely related to the inhibitory events

on the electron donor side of PSII linked to the oxygen

evolution complex. This new step corresponds to the

176 Plant Growth Regul (2013) 70:175–185

123

response to stress conditions such as salinity, water deficit,

temperature and light (Strasser 1997; Kouril et al. 2001;

Lazar 2006). However, in some situations, the appearance

of step K can reflect the suppression of steps I and J, with

high variations in the amplitude of the fluorescence inten-

sity of step ‘‘P’’ (Strasser 1997; Misra et al. 2001; Bukhov

et al. 2003; Panda et al. 2006).

Studies performed in silica (Radloff et al. 1998) and

in vivo (Dubeau et al. 1998) show that sHSPs, including

the sMT-sHSP23.6, participate as a response and/or pro-

tection mechanism against abiotic stresses, raising the

hypothesis that tolerance to heat stress could be increased

by either maintaining or increasing the expression of

the genes encoding this protein. In the following study,

the photosynthetic activity was analysed to determine the

effect of high temperature cycles on genotypes of tomato

‘Micro-Tom’ plants with different expressions of mito-

chondrial sHSP23.6 (MT-sHSP23.6).

Materials and methods

Plant material and experimental conditions

Tomato plants (Solanum lycopersicum Mill. cv. Micro-

Tom) were cultivated under a 12 h photoperiod with a light

intensity of 220–250 lmol m-2 s-1, at 25/20 �C day/night

temperature and a relative humidity of 75–80 %. The seeds

of non-transformed plants were disinfested by immersing

for 15 min in sodium hypochlorite solution (300 mg L-1)

at pH 6.0 containing 0.1 % Tween-20 and rinsed seven

times with distilled sterilised water. The seeds were

allowed to dry on Whatman no. 3 filter paper and placed in

recipients containing 50 mL half-strength Murashige and

Skoog (MS) culture medium at pH 5.9 enriched with

R3 vitamins (0.5 mg L-1 thiamine, 0.25 mg L-1 nicotinic

acid and 0.5 mg L-1 pyridoxine), 1.5 % (w/v) sucrose and

0.8 % (w/v) agar. To produce transgenic plants, a complete

fragment of cDNA (618 bp, gi/15234969) was cloned into

the binary vector pGA643 in the sense and antisense ori-

entations under the transcriptional control of the cauli-

flower mosaic virus 35S promoter and the nopaline

synthase terminator. The transformation was obtained

through the Agrobacterium tumefaciens system using 4- to

6-day-old cotyledons. The duration of subcultures for the

formation of the aerial parts was 15 days. Kanamycin was

used at a concentration of 100 mg L-1. The selection of

transformed plants was performed by growing them on

kanamycin and by PCR. The seeds of the F-3 generation

of self-pollinated plants grown in 100 mg L-1 kanamycin

were used. The genotypes were denominated as ‘‘WT’’ for

non-transformed plants, ‘‘S’’ for plants transformed in the

sense orientation and ‘‘AS’’ for plants transformed in the

anti-sense orientation for normal, high and low expression,

respectively, of MT-sHSP23.6. The MT-sHSP23.6 mRNA

and protein were detected by quantitative PCR (qPCR) and

Western blot, respectively.

The seeds of genotypes WT, S and AS were allowed to

germinate separately in plastic boxes in the dark at 24 �C,

and after 8 days, the seedlings were transplanted to washed

sand in plastic pots of 0.5 kg capacity. The plants were

raised in a growth chamber at 21 ± 2 �C, with a 12 h

photoperiod and photosynthetically active radiation of

approximately 200 lmol photons m-2 s-1. The plants

periodically were fertilised with nutrient solution (Hoa-

gland and Arnon 1950). The 59-day-old plants, still com-

pletely in the vegetative stage, were subjected to the first

cycle of high temperature stress for 24 h at 37 ± 1 �C and

then returned to the original temperature for 24 h for

recovery. This temperature cycle was followed by a second

cycle to verify if the effects are cumulative. The stress was

applied in growth chambers with a photoperiod of 12 h and

a photon flow density of approximately 50 lmol m-2 s-1

of photosynthetically active radiation. The parameters of

transient Chl a fluorescence and of gas exchange were

evaluated after each stress and recovery cycle.

Quantitative real-time PCR (q-PCR) and western blot

RNA was extracted from leaves following the protocol

described for PureLinKTM reagent (Plant RNA Reagent—

InvitrogenTM). The quantity and quality of the RNA was

assessed spectroscopically, electrophoretically in agarose

gel and chromatographically. The total RNA was treated

with DNase I (InvitrogenTM), and each sample was reverse

transcribed into cDNA using the commercial kit Super-

Script First-Strand System for RT-PCR (InvitrogenTM).

MT-sHSP23,6 gene-specific primers (Forward 50-CAAT

GGCTCTAGCTCGTCTGGCTTT-30; Reverse 50-TTCAC

CAGCCGAAGTAGCCATGAAT-30) were designed from

sequences (gi/15234969) deposited in GenBank using

Vector NTI AdvanceTM 10. The size of the amplification

products and their specificity were tested in agarose gels

(2 %, w/v) prior to q-PCR. The q-PCR was performed with

a 7500 Real-Time PCR System (Applied Biosystems�)

using SYBR� Green. The amplification reaction was car-

ried out in a total volume of 25 lL, containing 2 lM of

each primer, 12.5 lL PCR Master Mix SYBR� Green,

1 lL cDNA (diluted fivefold) and water to make up

the final volume. The samples were loaded in 96-well optic

plates (Applied Biosystems�) and covered with optic

adhesives (Applied Biosystems�). The relative quantifica-

tion of expression was performed using the comparative

threshold cycle method, as described by Livak and

Schmittgen (2001). For each cDNA, 18S (Forward 50-AA

AACGACTCTCGGCAACGGATA-30; Reverse 50-ATGG

Plant Growth Regul (2013) 70:175–185 177

123

TTCACGGGATTCTGCAATT-30) and b-actin (Forward

50-CGTCTTCCCCTCCATCG-30; Reverse 50-CTCGTTA

ATGTCACGCAC-30) were used as reference genes to

quantify cDNA abundance. The cycle threshold (CT) was

calculated based on the PCR exponential reaction obtained

from the relative expression level (REL) formula,

REL = 2-DDCT. The mRNA abundance of WT leaves

served as the baseline for determining the relative RNA

levels.

The immunodetection of proteins was performed by

Western blotting using mouse polyclonal antibodies pro-

duced against the recombinant MT-sHSP23.6 protein. To

this end, full-length MT-sHSP23,6 cDNA was cloned using

the vector pCR2.1-TOPO-TA (Invitrogen�) and transferred

to the expression vector pAE. The cloning was performed in

duplicate, and the clones were resequenced prior to Esche-

richia coli BL21 pLyss transformation. The recombinant

proteins were affinity-purified on a HisTrap column AKTA

Prime (Amersham Biosciences�) and then inoculated into a

mouse to obtain the polyclonal antibodies. To monitor the

MT-sHSP23.6 expression, proteins were extracted from

mitochondria, chloroplast and tomato leaves. The isolation

of mitochondria from tomato leaves was performed essen-

tially as described by Millar et al. (2001, 2004), who opti-

mised the protocol for Arabidopsis and rice. The chloroplast

fraction was also prepared from transformed sense MT-

sHSP23.6 tomato leaves, following the exact procedure

described by Barsan et al. (2010). Fractions containing

mitochondria or chloroplasts were diluted 1:10 in a buffer

made of 0.3 mannitol, 2 mM EDTA, and 10 mM TES (pH

7.5). After two centrifugation steps of 15 min at 15,0009g,

the pellet was dissolved in Laemmli buffer and stored at

-20 �C. The SDS-PAGE was performed by loading 1.0 lg

total mitochondrial or chloroplastic proteins. The total pro-

teins were extracted from 3.0 g frozen leaves ground into

powder and extracted with a solution containing 1.5 mL

280 mM Tris–HCl (pH 8.3), 0.5 M DTT, 20 % v/v glycerol

and 4 % w/v SDS. b-mercaptoethanol (10 %, v/v) was added

just before heating the samples at 80 �C for 10 min. The

samples were then cooled at 4 �C and centrifuged at

14,0009g for 30 min. Protein quantification was performed

according to the Bradford (1976) method following precip-

itation with trichloroacetic acid and solubilisation with

0.1 M sodium hydroxide. Equal amounts of total protein

(30 lg) were loaded per gel slot onto a denaturing SDS-

PAGE gel. The proteins were transferred from the poly-

acrylamide gel to a nitrocellulose membrane (Hybond ECL,

GE Healthcare�), and the efficacy of the transfer was mon-

itored by colour using Ponceau’s reagent. The membrane

was blocked with 2 % (w/v) ECL Advance Blocking� agent

in a Tris-buffered saline Tween solution [TBS-T, 20 mM

Tris, 137 mM NaCl and 0.1 % (v/v) Tween-20, pH 7.6] for

1 h at room temperature (RT) with agitation. The anti-MT-

sHSP23,6 antibody was diluted in TBS-T 1:1,000 and

incubated for 1 h at RT. The membranes were washed three

times for 10 min with TBS-T and incubated for 1 h at RT

with a 1:50,000 dilution of the peroxidase-labelled rabbit

anti-mouse antibody (GE Healthcare�). The membranes

were then washed with water and developed using the GE

Healthcare� Kit (ECL Advance Western� blotting detection

reagents), with an exposure time of approximately 30 s.

Chl a fluorescence transient induction

Chl a fluorescence transient induction (OJIP curve) was

measured in the first fully expanded young leaves with the

use of a portable fluorometer (Model Handy PEA, Hansa-

tech Instruments, King’s Lynn, Norfolk, UK.). The mea-

surements were taken on leaves in situ previously adapted

to the dark for 30 min. The measurement consisted of

a single strong 1 s light pulse (3,000 lmol m-2 s-1, an

excitation intensity sufficient to ensure closure of all PSII

reaction centres) provided by an array of six light-emitting

diodes (peak 650 nm). The Chl a fluorescence emission

induced by the strong light pulses was measured and

digitised between 50 ls and 1 s by the instrument. The

fluorescence intensities were determined at 50 ls (F50ls),

100 ls (F100ls), 300 ls (F300ls), 2 ms (F2ms) and 30 ms

(F30ms) and Fm (maximum fluorescence). The intensity

measured at 50 ls was considered the initial fluorescence

(F0). The measured fluorescence intensities were used to

calculate the parameters established by the JIP-test (Strasser

and Strasser 1995) with the use of the ‘Biolyzer’ software

(Laboratory of Bioenergetics, University of Geneva,

Switzerland, supplied by Dr. R. Strasser).

Gas exchange

The gas exchange was measured in the top-most fully

expanded leaves with the use of a portable infra-red CO2

analyser (model LI-6400XT LI-COR, Inc., Lincoln, NE,

USA). The measurements were taken between 10:00

and 11:00 a.m., with an in-chamber CO2 concentration

of 380 mol mol-1 and a photon flow density of 1,500

lmol m-2 s-1, using the light source LI-COR 6400-02

attached to the measuring chamber.

Experimental design and statistical analysis

All experiments were arranged in a completely randomised

design with five replications. The fluorescence transient

OJIP was measured three times per replication, with a

total of 15 measurements per treatment. The fluorescence

parameters were normalised relative to the pre-stress

measurements (control).

178 Plant Growth Regul (2013) 70:175–185

123

Results

Transcriptional and transductional expression of

MT-sHSP23.6

To validate the experimental model, the relative accumu-

lation of transcripts and transduction products was mea-

sured in the leaves of WT, AS and S plants. The effective

genetic transformation occurred with the clone MT-

sHSP23.6 anti-clockwise, which reduced the relative

accumulation of transcripts of this gene; mRNAs were not

detected (Fig. 1). In contrast, in S plants transformed for

high expression of this gene, the relative accumulation of

the transcripts was 759 higher than in the control plants

(Fig. 1a).

A high immunoreactivity was detected in the mito-

chondrial enriched fraction prepared from transformed

tomato leaves overexpressing this gene (S-plants) (Fig. 1b).

No immunoreactions were detected in the AS plant proteins

and chloroplastic proteins. This immunoreactivity indicates

that the MT-sHSP23.6 protein is targeted to mitochondria

(Fig. 1b). This finding is important because the MT-

sHSP23.6 protein is predicted to be dual targeted by

iPSORT II Service (Chl and Mt) Mitoprot (Mt), ChloroP 1.1

Server (Chl), TargetP 1.1 Server (Mt), and Predotar (Mt). In

the leaves of S plants, the intensity of the corresponding

immunodetected band was greater than in the control plants,

while this protein was not detected in AS plants. These

results indicate a model in which the expression of the MT-

sHSP 23.6 gene is silenced because the respective mRNAs

and corresponding proteins were not detected. In contrast,

the transcript accumulation of the MT-sHSP23.6 protein

was increased in the S plant (Fig. 1).

Chl a Fluorescence transient: JIP-test, normalisation and

subtraction of relative variable fluorescence

The analysis of the fluorescence transient according to the

JIP-test leads to the calculation of several phenomenolog-

ical and biophysical expressions (Tsimilli-Michael and

Strasser 2008). The methodological framework of the

energy flux theory permits the rigorous definition of all

terms often used in the analysis of energy distribution in

the photosynthetic apparatus (biophysical parameters, flux,

yield, probability and rate constants of excitation energy

transfer) (Strasser et al. 2004). The translation by the JIP-

test of original data from a fluorescence transient to the

biophysical parameters that quantify the PS II behaviour is

summarised in Strasser et al. (2004). These authors indicate

that many of the JIP-test parameters are interdependent and

that they are derived from the O-J phase, hence providing

information for QA reduction by single turnover events,

and a group of other parameters provide information for QA

reduction by multiple turnover events (Strasser et al. 2004).

The total complementary area normalised above the

transient OJIP (Sm) curve increased in WT and S plants but

not in AS plants. However, the total complementary area

normalised above the curve of only the transient OJ(Ss)

phase, which reflects the events of simple cycle QA

reduction, was not affected by heat stress in any genotype.

The turnover number as reduction, oxidation, and

re-reduction of QA in the timespan from the light on until

reaching FM (N) increased in WT and S plants and was

more accentuated during the second stress cycle in WT

plants (Fig. 2).

The specific flux per reaction centre (RC), the absorp-

tion flux (ABS/RC), the trapped energy flux (TR0/RC) and

the electron transport flux (ET0/RC) increased relative to

the WT plants after two stress cycles but returned to values

close to the control in all genotypes (Fig. 2). However, the

electron flux reducing end electron acceptors at the PSI

acceptor side per RC (RE0/RC) increased only in the

WT and S plants. The dissipation flux per RC (DI0/RC)

increased the most in response to heat stress in all

Fig. 1 Relative transcript accumulation of the MTsHSP23.6 gene in

tomato leaves (a) and protein immunodetection of the MT-sHSP23.6

gene (b) from 1—chloroplastic fraction from S plants; 2, 3, 4—

mitochondrial fractions from AS, WT and S plants, respectively; 5, 6,

7—crude proteins extract from AS, WT and S plants, respectively

from tomato leaves. AS antisense line, WT wild type, S sense line

Plant Growth Regul (2013) 70:175–185 179

123

genotypes, which returned to values similar to the control

only after the first recovery phase but remained high after

the second recovery phase.

The maximum quantum yield for primary photochem-

istry (TR0/ABS = FV/FM = uP0) and quantum yield for

electron transport (uEo = ET0/ABS) did not differ among

the treatments. In contrast, in WT and S plants, the quan-

tum yield for the reduction of end acceptors at the PSI

acceptor side (uRo = RE0/ABS) increased after the first

stress phase (Fig. 2).

In all genotypes, the performance index (potential for

energy conservation from photons absorbed by PSII to the

reduction of intersystem electron acceptors—PIABS)

decreased across the stress cycles and did not return to

levels similar to the controls after the recovery phase

(Fig. 2). However, the performance index decreased from

photons absorbed by PSII to the reduction of PSI end

acceptors (PItotal) (Tsimilli-Michael and Strasser 2008) in

AS plants but increased in the WT and S plants.

The normalisation between steps O (50 ls) and K

(300 ls) represented a kinetic difference [DWOK = (WOK

(treatment) - WOK (control))] (Fig. 3a–c), showed the presence

of a positive L-band (at approximately 150 ls) in all

genotypes. This band indicates the connectivity or group-

ing of PSII units, which, if positive, indicates a low con-

nectivity (Yusuf et al. 2010).

The normalised data between steps O (50 ls) and J

(2 ls) represent as relative variable fluorescence and are

described as the kinetic difference [DWOJ = (WOJ (treatment)

- WOJ (control))] (Fig. 3d–f), permitting the visualisation of

a K band (*300 ls), which, if positive, indicates the

inactivation of the oxygen evolution complex (Yusuf et al.

2010). This band was positive in all genotypes immediately

after the heat stress and after the first recovery phase.

To identify the effect of high temperature on the O–I

phase of the fluorescence transient OJIP, the curves were

normalised as the relative variable fluorescence between

steps O and I [WOI = (Ft - F0)/(FI - F0)] (Fig. 4a–c). The

analysis of the variable fluorescence between steps O and I

is less than 1 (WOI \ 1), permitting the evaluation of the

sequence of events from trapping by the RC of PSII until

the reduction of the plastoquinone. All genotypes showed a

low WOI after the recovery phase (Fig. 4a–c), which indi-

cates a low oxidation rate of the final electron acceptors on

the acceptor side of PSII.

The I–P phase was evaluated by two distinct procedures:

(1) interpretation of the normalisation of variable fluores-

cence between steps O and I, with values greater than or

equal to 1 (WOI C 1) (Fig. 4d–f). This interpretation permits

the evaluation of the sequence of events of electron transfer

from the reduced plastoquinone to the final electron acceptor

of PSI. (2) The normalisation of fluorescence transient data

between steps I and P (WIP = [Ft - FI]/[FM - FI]) on a

linear scale between 30 and 300 ms (Fig. 4g–i).

The WOI C 1 (Fig. 4d–f) reflects the pool size of elec-

tron acceptors on the electron acceptor side of PSI. There

was a marked difference among the genotypes in the O–I

phase, with the greatest difference being between WT and

S plants, which, after each stress phase, showed a greater

amplitude of curves, indicating a larger pool of electron

acceptors on the electron acceptor side of PSI. However,

the values were similar to the control immediately after

recovery. This phenomenon was not observed in AS plants.

(a)

(b)

(c)

Fig. 2 Radar plot of several parameters calculated by the JIP-test of

the tomato plants ‘Micro-Tom’ with different expressions of HSP

after high temperature stress (24 h, 37 �C). a Wild type, b high

expression MT-sHSP23.6, c low expression MT-sHSP23.6. (Solidline) control, (filled square) after first stress, (filled circle) after first

recovery, (open square) after second stress and (open circle) after

second recovery. The heavy black line represents the normalised

values of the parameters of the control (non-stressed -21 �C)

180 Plant Growth Regul (2013) 70:175–185

123

Gas exchange

The net assimilation of CO2 (A) (Fig. 5) increased after two

heat stress cycles in WT and S plants; however, in the latter

plants, the increase was more marked, especially after the

second cycle. On the other hand, in AS plants, the net

assimilation of CO2 decreased after the first cycle. Although

the net assimilation of CO2 increased after the first recovery

phase, it always remained below that of the S plants.

Discussion

One of the mechanisms involved in acquiring heat toler-

ance is the induction of HSP codifying genes (Hajek et al.

2005). This study reports the relationship between the MT-

sHSP23.6 gene that codifies a protein found in mitochon-

dria and in heat stress tolerance. Although photosynthesis

is highly affected by heat stress, it is hypothesised that the

stability of the endomembrane system and other organelles

can affect the photosynthetic metabolism.

Whereas the mitochondrial plant genome is very poor

and contains approximately 100 protein-coding genes, the

mitochondrial plant proteome is considered complex and

consists of approximately 2,000–3,000 proteins (Millar

et al. 2001, 2004, 2005, 2008). Therefore, the large

majority of mitochondrial proteins are encoded by the

nuclear genome and imported into the organelle. Among

1196 Arabidopsis thaliana genes that putatively encode

mitochondrial proteins, 45 nuclear-encoded genes were

characterised as widely stress-responsive, including MT-

sHSP23.6 (Van Aken et al. 2009). More specifically, MT-

sHSP23.6 transcript accumulation was observed when

A. thaliana was submitted to heat, UV or H2O2 (Rizhsky

et al. 2004; Charng et al. 2007; Van Aken et al. 2009).

However, these results are not sufficient to prove that this

gene is involved in abiotic stress tolerance. No work has

been performed correlating changes in MT-sHSP23.6

expression and abiotic stress tolerance. Thus, we produced

mutants with high and low MT-sHSP23.6 levels to test

this hypothesis. By genetic transformation, it was effec-

tively verified that this protein is targeted to mitochondria

(Fig. 1b) and is involved in heat tolerance.

In the transformed plants in which the gene expression

was suppressed, the mRNAs and the corresponding pro-

teins were not detected, and in S plants in which the syn-

thesis was induced, there was a 759 greater increase in the

transcripts and a higher density of the protein band. Thus,

Fig. 3 The difference in the kinetics of double normalisation at F0

and FK [DWOK = WOK [treatment] - WOK [control]] (a–c) and the

difference in the kinetics of double normalisation at F0 and FJ

[DWOJ = WOJ [treatment] - WOJ [control]] (d–f) to the tomato plants

‘Micro-Tom’ with different expressions of HSP after high temper-

ature stress (24 h, 37 �C). a, d—WT, b, e—high expression

MT-sHSP23.6, c, f—low expression MT-sHSP23.6

Plant Growth Regul (2013) 70:175–185 181

123

this model was considered suitable for this study because it

allowed plants with non-detectable levels of MT-sHSP23.6

mRNA or protein (AS), as well as low (WT) and high

(S) levels. High temperature stress, associated or not with

other stress agents, is considered a major factor affecting

plant growth and development, due to the high sensitivity

of the photosynthetic apparatus. Nautiyal and Shono (2010)

correlated the photosynthetic thermotolerance with the

production of sHSP from transgenic tomatoes Mt-sHSP

(for mitochondrial proteins) and ER-sHSP (proteins in the

Fig. 4 Double normalisation at the F0 to FI [WOI = (Ft - F0)/(FI -

F0)] (a, f) and double normalisation at the FI to FP [WIP = (Ft - FI)/

(FM - FI)] (g, i) to the tomato plants ‘Micro-Tom’ with different

expression levels of HSP after high temperature stress (24 h, 37 �C).

a, d, g—WT, b, e, g—high expression MT-sHSP23.6, c, f, i—low

expression MT-sHSP23.6. Individual events starting from the photons

absorbed (ABS) followed by exciton trapping (TR), electron transport

(ET), and plastoquinone (PQ) reduction (O–I phase, from 0 to 1) by

the PSII; then from the PSI-driven electron transport to the reduction

of end electron acceptors (RE) on the PSI acceptor region that started

at PQH2 to Cyt bf complex to PC. d–f Events for WOI C 1.0 illustrate

the differences in the pool size of the end electron acceptors.

g, h Comparison of the reduction rates in the pool of the end electron

acceptors

182 Plant Growth Regul (2013) 70:175–185

123

endoplasmic reticulum), and they observed that the Mt-

sHSP were more beneficial than ER-sHSP because the

Mt-sHSP were able to provide thermotolerance to maintain

a stable net photosynthesis and to preserve the stability of

the cell membrane. Wahid et al. (2007) emphasise that PSII

is highly thermolabile and that its activity is considerably

reduced and even partially interrupted under high temper-

atures. It is also hypothesised that heat stress can lead to the

disassociation of the OEC, resulting in a disequilibrium

between the electron transport of OEC in the direction of

the acceptor side of PSII to the direction of the RC of PSI.

This disequilibrium was found in this study, as indicated by

the presence of the K band (Fig. 3).

The tomato plants subjected to 37 �C showed alterations

in parameters of Chl fluorescence, especially in genotypes

with high MT-sHSP23.6 (S) and WT plants. These geno-

types showed an increase in parameters related to the activity

of the electron acceptors of PSII, as well as an elevated

capacity of absorption, trapping and energy transfer, result-

ing in the stability of the performance indexes relative to the

control plants.

The PItotal values of S and WT plants corroborate with

those of Yusuf et al. (2010), who showed that the increase

of PItotal over the control is indicative of a positive tension

on the system, which clearly demonstrates that the induced

structural modifications allow a better performance of the

photosynthetic machinery under heat stress. This fact is

related to the presence of HSPs, which normally are

associated with cellular structures such as the cell wall,

chloroplasts, mitochondria and ribosomes. In tomato plants

under heat stress, the HSPs aggregate as a granular struc-

ture in the cytoplasm, most likely to protect the protein

biosynthesis machinery or to avoid protein denaturing by

high temperature (Wahid et al. 2007). There are also

reports suggesting that high molecular weight HSPs have

basic cellular functions, even in the absence of stress, while

the low molecular weight HSPs are directly involved in

survival and heat stress recovery, as well as specific

developmental processes (Waters et al. 1996), which could

justify the better photosynthetic performance of WT plants

and high MT-sHSP23.6 plants.

The presence of L and K bands and the normalisation

between steps O and K and O and J, respectively, permit

the comprehension of several aspects, allowing for a more

plausible and precise approach regarding the effect of heat

stress on the electron transport chain (ETC) of photosyn-

thesis. The positive bands indicate deleterious effects on

plants. As the HSPs are involved in protecting the photo-

synthetic machinery, especially the PSII, against the dam-

age caused by stresses that induce photo-oxidation or by

heat stress (Lee et al. 2000), it can be assumed that the

presence of these proteins could be related to cell protec-

tion, to assure functioning of vital processes, especially

those involved in energy production during the period in

which plants are exposed to high temperature (Lin et al.

1984). This relationship was proved in this study.

The higher quantum yield for reduction of the final

electron acceptors, as observed in S and WT plants

(Fig. 2), could be associated with the proposal of Ogweno

et al. (2009), which suggests that there must be other

electrons sinks in the ETC and that heat induces a cyclic

stimulation of electron transport around PSI that could be

responsible for the increase of pH and of the Mehler

reaction. The AS plants lost energy along the ETC, and

because there was no significant yield at the end of the flux,

relative to other genotypes, it is possible that this energy

was used for repairs along the chain. Nautiyal et al. (2005)

report that the HSPs protect the NADH:ubiquinone oxi-

doreductase complex of ETC during heat stress.

Considering the net assimilatory rate, the S plants showed

the greatest adaptation to heat stress, which confirms the

observation of Heckathorn et al. (1998) that indicates that the

HSPs constitute an important component in acquiring heat

tolerance. Their expression and association with mitochondria

protect them from oxidative phosphorylation when the

mitochondria are subjected to high temperatures. Chou et al.

(1989), in studies with soybean, found a correlation between

the maintenance of oxidative phosphorylation and the presence

of HSPs between 15 and 30 kDa, even when mitochondria

were subjected to 42.5 �C. Similarly, in C4 plants (sorghum,

millet and Urochloa panicoides L.), Clarke and Critchley

(1990) found the inhibition of protein synthesis by treatment

with cycloheximide during heat shock, which caused a drastic

Fig. 5 Net assimilation of CO2 (lmol CO2 m-2 s-1) by the tomato

plants ‘Micro-Tom’ with different expression levels of HSP after high

temperature stress (24 h, 37 �C) (Bar—SE of mean) (n = 5)

Plant Growth Regul (2013) 70:175–185 183

123

reduction in the efficiency of photosynthesis, thus suggesting

that HSP synthesis has a protective role in chloroplasts.

The net assimilation of CO2 was higher in WT and S

plants after heat stress cycles. This fact not was observed in

AS plants, suggesting that the lower expression of mito-

chondrial sHSP23.6 provoked a decline in photosynthesis,

as these proteins protect photosynthesis from high tem-

perature stress (Fig. 5). Mohanty et al. (2012) show that the

primary targets of high temperature stress in plants are the

oxygen evolution complex and its associated cofactors in

PSII, carbon fixation by Rubisco and the ATP generating

system, and the induction of the expression of stress pro-

teins that alleviate the ROS-mediated inhibition of stress

damage repair of the photosynthetic machinery and are

required for the acclimation process.

According to Wahid et al. (2007), plant growth reduc-

tion is one of the main consequences of stress. This

reduction occurs especially due to a reduction in the net

photosynthetic rate, generation of reductive power, and

interference with mitochondrial functions. It has been

suggested that during photoreactions, the increase of leaf

temperature induces the synthesis of ATP to equilibrate

ATP consumption under heat stress, possibly by the cyclic

electron flow (Bukhov et al. 1999). During the ‘‘dark

reaction’’ of photosynthesis in the Calvin cycle, the acti-

vation of Rubisco is considered a critical step that can be

inhibited at 35–40 �C, leading to a net decrease in CO2

assimilation and carbohydrate production. The optimum

temperature for tomato cultivation lies between 25 and

30 �C during the photoperiod and 20 �C during the dark

period (Camejo et al. 2005). Thus, in response to high

temperature stress, the high expression of mitochondrial

sHSP23.6 results in less damage to the photosynthetic

activity, suggesting that some degree of heat tolerance can

be attributed to these proteins. Therefore, this fact suggests

the potential utility of breeding, engineering, or selecting

plants for increased production of mitochondrial sHSP23.6

in improving plant tolerance to stress. Of course, the

increased production of the mitochondrial sHSP23.6 in

response to high temperature stress is only one of the many

plant adaptive responses to this stress.

Acknowledgments We acknowledge the financial support of the

CNPq (Conselho Nacional de Desenvolvimento Cientıfico e Tec-

nologico), Ministerio de Ciencia e Tecnologia, FINEP, CAPES

(Coordenacao de Aperfeicoamento de pessoal de Nıvel Superior), and

FAPERGS (Fundacao de Amparo a Pesquisa do Estudo do Rio

Grande do Sul).

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