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Coordinate changes in photosynthesis, sugar accumulationand antioxidative enzymes improve the performance ofJatropha curcas plants under drought stress

Evandro N. Silva a, Rafael V. Ribeiro b, Sergio L. Ferreira-Silva a, Suyanne A. Vieira a,Luiz F.A. Ponte c, Joaquim A.G. Silveira a,*a Laboratorio de Metabolismo de Plantas, Departamento de Bioquımica e Biologia Molecular, Universidade Federal do Ceara,

CP 6004, CEP 60451-970, Fortaleza, Ceara, Brazilb Laboratorio de Fisiologia Vegetal ‘Coaracy M. Franco’, Instituto Agronomico, CP 28, CEP 13012-970, Campinas, Sao Paulo, BrazilcUniversidade Estadual Vale do Acarau, Centro de Ciencias Agrarias e Biologicas, CEP 62040-370, Sobral-CE, Brazil

a r t i c l e i n f o

Article history:

Received 29 November 2010

Received in revised form

5 June 2012

Accepted 7 June 2012

Available online 30 June 2012

Keywords:

Antioxidant enzymes

Carbohydrates

Jatropha curcas

Photochemical activity

Photorespiration

Water stress

* Corresponding author. Tel./fax: þ55 853366E-mail addresses: joaquim.silveira@pesqu

0961-9534/$ e see front matter ª 2012 Elsevhttp://dx.doi.org/10.1016/j.biombioe.2012.06.0

a b s t r a c t

The aim of this study was to assess the relationships between photosynthesis, sugars and

photo-oxidative protection mechanisms in Jatropha curcas under drought stress. Leaf CO2

assimilation rate (PN) and instantaneous carboxylation efficiency decreased progressively

as the water deficit increased. The sucrose and reducing sugar concentrations were

negatively and highly correlated with photosynthesis indicating a modulation by negative

feedback mechanism. The alternative electron sinks (ETRs’/PN), relative excess of light

energy (EXC) and non-photochemical quenching were strongly increased by drought,

indicating effective mechanisms of energy excess dissipation. The photochemistry data

indicate partial preservation of photosystem II integrity and function even under severe

drought. EXC was positively correlated with superoxide dismutase (SOD) and ascorbate

peroxidase (APX) activities evidencing an effective role of these enzymes in the oxidative

protection against excess of reactive oxygen species in chloroplasts. Leaf H2O2 content and

lipid peroxidation were inversely and highly correlated with catalase (CAT) activity indi-

cating that drought-induced inhibition of this enzyme might have allowed oxidative

damage. Our data suggest that drought triggers a coordinate down-regulation in photo-

synthesis through sucrose and reducing sugar accumulation and an energy excess dissi-

pation at PSII level by non-photochemical mechanisms associate with enhancement in

photorespiration, restricting photo-damages. In parallel, drought up-regulates SOD and

APX activities avoiding accumulation of reactive oxygen species, while CAT activity is not

able to avoid H2O2 accumulation in drought-stressed J. curcas leaves.

ª 2012 Elsevier Ltd. All rights reserved.

1. Introduction general interest to produce biodiesel from Jatropha curcas

Biodiesel is an alternative to petroleum diesel fuel. It is

a renewable, a biodegradable, and also a non-toxic fuel. The

9821.isador.cnpq.br, silveira@ier Ltd. All rights reserve09

seeds oil has increased but its ability to grown on drought-

prone areas has scarcely been studied [1]. J. curcas is distrib-

uted over the arid and semi-arid areas of South America and

ufc.br (J.A.G. Silveira).d.

b i om a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 7 0e2 7 9 271

in all tropical regions. This species n the last years it has

recently received tremendous attention because its high seed

oil contentwhich can be converted to biodiesel. Accordingly, it

is being considered as a universally accepted energy source

crop [2]. This species grows in areas with extreme climates

and soil conditions that could not be habited by most of the

agriculturally important plant species [3].

Water availability is an essential factor affecting plant

growth and yield, especially in arid and semi-arid regions,

where plants are often subjected to long periods of drought.

Morphological and physiological responses to drought stress

may vary considerably among plant species, causing differ-

ential performance under water-limiting conditions. In

general, strategies of drought avoidance or drought tolerance

can be recognized, both involving diverse physiological and

biochemical mechanisms that enable a plant to grow and

survive under drought conditions [4].

Photosynthesis is one of the most sensitive processes to

drought stress [5,6]. The inhibitory effects of drought on

photosynthesis may be associated with low CO2 availability

due to low stomatal and mesophyll conductances [7] and/or

impairments in carbon assimilation metabolism [8]. Stomatal

closure is an early response to drought and an efficient way to

reduce water loss in water-limiting environments. Biochem-

ical limitation of photosynthesis also plays an important role

under prolonged periods of drought stress [7]. The accumu-

lation of sugars in drought-stressed leaves might exert

a modulation on photosynthesis by a negative feedback

mechanism [9]. The biochemical limitation of photosynthesis

under drought stress has been associated with low carboxyl-

ation efficiency due to reducedmaximum rate of ribulose-1,5-

bisphosphate (RuBP) carboxylation and low regeneration rate

of RuBP driven by the electron transport rate [10]. The effects

of water stress on Rubisco activity vary with plant species and

the level of drought stress, ranging from a dramatic reduction

[11] to little or no inhibition of the enzyme [12]. Some studies

have found that photosynthesis is not inhibited by Rubisco

activity until severe drought stress is reached [13]. Under

severe water stress, impaired photochemistry may also limit

photosynthesis [14].

Water stress can also induce oxidative damage in leaves

due to an imbalance between the light capture and electron

utilization in CO2 fixation [6]. Under such conditions, leaves

face an increase in the generation of reactive oxygen species

(ROS), with oxidative damage arising when the accumulation

of ROS exceeds the removing capacity of the antioxidant

systems, mainly in chloroplasts and peroxisomes. The

consequences of oxidative stress include peroxidation of

membrane lipids, degradation of photosynthetic pigments

and inactivation of photosynthetic enzymes [6]. Many plant

species have evolved multiple photoprotective and antioxi-

dant mechanisms to withstand drought-induced oxidative

stress. Apart from the xanthophyll cycle, photorespiration

and other changes in metabolic activity, a number of enzy-

matic (e.g., SOD, APX and CAT) and non-enzymatic (e.g.,

ascorbate and glutathione) antioxidants found in chloroplasts

and peroxisomes serve to prevent ROS accumulation and

consequent oxidative damage [15]. Moreover, recent experi-

mental evidences have shown the cytosolic APX activity is

essential to chloroplast protection against oxidative damage

[16]. Furthermore, soluble sugars, such as glucose and sucrose,

are now recognized as crucial compounds in coordinating

plant developmental responses under oxidative stress [15].

Oxidative stress in leaves depends on the balance between

photochemistry, CO2 assimilation, photorespiration and

antioxidant mechanisms. Among the antioxidative enzymes,

SOD plays an essential role in the protection of chloroplasts

against oxidative damage, converting O$�2 to H2O2. The

hydrogen peroxide is then eliminated by H2O2-scavenging

enzymes, such as peroxidases and catalases. APX is widely

distributed in the cytosol and in other plant organelles and is

very effective in scavenging H2O2 generated by SOD through

the waterewater cycle, mainly in chloroplasts where CAT is

virtually absent [17]. The thylakoid-APX isoform can be inac-

tivated by excess H2O2 generates in the chloroplasts which

can occur under stressful conditions such as water deficit [17].

Although the photosynthetic process has been widely

studied under drought conditions, little is known about the

regulatory role exerted by sugars [18]. Indeed, an excess of

sugars might repress photosynthetic gene expression, while

deprivation of sucrose and glucose might stimulate photo-

synthesis [19]. Moreover, little is also known about the rela-

tionships between photochemical and biochemical activities

associated with CO2 assimilation under drought stress. An

imbalance between photochemical reactions and Rubisco

carboxylase activity can produce an excess of energy, which

can up-regulate photorespiration and then dissipate excess

energy and/or generate reactive oxygen species in excess in

chloroplasts and peroxisomes [5,20].

We have recently demonstrated that young J. curcas plants

exhibit an efficient osmotic adjustment in response to drought

by accumulating soluble sugars as their major osmolytes [21].

In addition, we have shown that drought stress combined

with high temperature significantly impairs photochemistry

and CO2 assimilation rates [22]. However, the roles of photo-

chemical and antioxidant mechanisms involved in the water

stress tolerance and photo-oxidative protection of J. curcas and

other plant species are not well known.

In this study, we tested the hypothesis that J. curcas plants

avoid severe photochemical damage under drought-stress

conditions through coordinate changes involving photosyn-

thetic CO2 assimilation, photochemical activity, carbohydrate

metabolism and an efficient antioxidant enzymatic system in

chloroplasts, consisting of SOD and APX activities and the

effective scavenging of excess H2O2 produced from photo-

respiration by CAT. We discuss the effectiveness of such

coordinated responses for drought tolerance.

2. Material and methods

2.1. Plant material and growth conditions

The initial phase of the experiment was carried out under

greenhouse conditions (3�440S; 38�330W, at sea level) during

January 2009. Seeds of J. curcas L., cultivar FT1, supplied by

Tamandua Farm, Santa Terezinha, Paraıba (Brazil) previously

selected for size and weight were surface sterilized for 1 min

with a sodium hypochlorite solution 5% (v v�1) and germi-

nated in pots (0.011 m3) filled with sand. Eight days after

b i om a s s an d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 7 0e2 7 9272

germination, seedlings of similar height and morphological

aspects were transplanted to pots (0.002 m3) filled with

medium-textured vermiculite and maintained in a water

volume fraction of 70% of vermiculite-holding capacity. Pots

were watered with half-strength Hoagland and Arnon [23]

solution every two days. The environmental conditions

inside greenhouse were: mean air temperature between 24 �C(minimum) and 36 �C (maximum) with an average tempera-

ture of 29 �C; average air relative humidity around 65%; the

maximum photosynthetic photon flux density (PPFD) of

500 mmol m�2 s�1 and 12 h-photoperiod. The plants were

grown under greenhouse conditions until they were 23 days

old (eight-leaf stage).

2.2. Water-deficit treatments and harvesting

The pots were transferred from the greenhouse to a growth

chamber with controlled environmental conditions: PPFD of

400 mmol m�2 s�1, air temperature of 27 �C, air relative

humidity of 70% and photoperiod of 12 h. To obtain a wide

range of water availability, water regimes from well-watered

to severely water-stressed conditions were imposed. The

water volume fraction for control plants was 70% of the

vermiculite-holding capacity (corresponding to a water mass

fraction of 50%), and the water deficit was progressively

increased by restricting irrigation in 0% (control treatment),

25%, 50%, 75% and 100%. This approach induced water-deficit

treatments with substrate water mass fraction of 50%, 40%,

30%, 20% and 10%. Plants were subjected to water-deficit

treatments for ten days. During the experiment, all pots

were weighed daily and irrigated to counterbalance water loss

with full-strength Hoagland and Arnon [23] solution. At the

end of the experiment, leaves were harvested, frozen and

stored at �80 �C for lyophilization and further chemical and

biochemical analyses.

2.3. Water status, electrolyte leakage, chlorophyllconcentration and leaf dry matter

The leaf water potential (Jw) was evaluated with a pressure

chamber at midday, using leaves similar to those used for leaf

gas exchange and chlorophyll-fluorescence measurements.

The leaf relative water content (RWC) and leaf succulence (LS)

were determined as previously described by Silva et al. [21].

For determining the osmolality, small segments from fully

expanded leaves were macerated with a mortar. After extract

filtration in amiraclothmembrane, the sapwas centrifuged at

10,000 g for 10 min at 4 �C. The resultant supernatant was

used to determine the osmolality (c) with a vapor pressure

osmometer (Vapro 5520, Wescor, USA). The osmotic potential

was determined using the formula: Js (MPa) ¼ �c

(10�3 � mol kg�1) � 2.58 � 10�3, according to the Van’t Hoff

equation.

Electrolyte leakage was assessed as described by Cav-

alcanti et al. [17]. Leaf discs were placed in closed tubes con-

taining 10�5 m3 of deionized water and incubated at 25 �C in

water bath for 6 h; subsequently electrical conductivity of the

solution (L1) was determined. Samples were then boiled at

100 �C for 1 h and a second electrical conductivity (L2) was

obtained after equilibration at 25 �C. The electrolyte leakage

(EL) was defined as follows: EL(%) ¼ (L1/L2) � 100.

The total chlorophyll content was calculated according

to Lichtenthaler [24]: Chl (g kg�1 FW) ¼ (7.15 � Abs663) þ(18.61 � Abs647) � V, where Abs is the absorbance and V the

final volume of the extract (10�6 m3). The total leaf dry matter

was evaluated after complete drying by lyophilization.

2.4. Leaf gas exchange and chlorophyll fluorescence

Leaf gas exchange was monitored with an infrared gas

analyzer (LCi, ADC, Hoddesdonm, UK), evaluating leaf CO2

assimilation rate (PN), stomatal conductance ( gS) and inter-

cellular CO2 concentration (CI). The chlorophyll fluorescence

was evaluated with a fluorometer (FMS 2, Hansatech, King’s

Lynn, UK). Minimum (FO), maximum (FM) and maximum

variable (FV ¼ FM�FO) fluorescence intensities were sampled

under steady-state conditions in dark-adapted (30 min)

leaves. In addition measurements were taken under in light-

adapted conditions, being referred as F0O (minimum) and F0M(maximum). The F0O signal was measured after PSI excitation

by far-red light. The fluorescence signal under light-adapted

conditions immediately before the saturation pulse is

referred as F0s and the variable fluorescence signal under light

conditions is DF0 ¼ F0M�F0s. The following photochemical vari-

ables were calculated: maximal (FV/FM) and actual (DF0/F0M)quantum yield of PSII, apparent electron transport rate

(ETRs’ ¼ DF0/F0M � PPFD � 0.5 � 0.84), and non-photochemical

[NPQ¼(FM�F0M)/(F0M)] quenching. For ETRs’ calculation, 0.5 was

used as the fraction of excitation energy distributed to PSII

and 0.84 as the fraction of incoming light absorbed by the

leaves. The relative excess of light energy (EXC) was calculated

according to Chagas et al. [25] as EXC ¼ [(FV/FM)e(DF0/F0M)]/(FV/FM). The instantaneous carboxylation efficiency (PN/CI) and

ETRs’/PN ratio were also calculated [20]. Leaf gas exchange and

chlorophyll fluorescence were measured at the same condi-

tions (27 �C and PPFD of 400 mmol m�2 s�1) in the same leaf.

Those measurements were taken after 10-d of water deficit

treatments.

2.5. Determination of carbohydrates

Lyophilized leaf samples were transferred to hermetically

closed tubes containing deionized water and placed in

a 100 �C water bath for 1 h. The total soluble sugar and

starch contents were determined using the phenol-sulfuric

method after hydrolysis of starch with perchloric acid [26].

Sucrose determinations were done by the anthrone method,

according to Van Handel [27]. Reducing sugar content was

estimated as the difference between total soluble sugars and

sucrose, while non-structural carbohydrate content was

quantified as the sum of total soluble sugar and starch

contents.

2.6. Hydrogen peroxide content and lipid peroxidation inleaves

Samples of fresh leaves (10�4 kg) were powdered in liquid

nitrogen and extracted with 0.1 mol m�3 potassium phos-

phate buffer (pH 6.4) containing 5 � 10�3 mol m�3 KCN,

b i om a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 7 0e2 7 9 273

according to Cheeseman [28]. The reaction was carried out at

25 �C for 30 min, and the absorbance was read at 560 nm. The

H2O2 concentration was calculated according to a standard

curve and expressed as 10�3 mol (kg FW)�1. Lipid peroxidation

was determined bymeasuring the thiobarbituric acid-reactive

substances (TBARS). For TBARS determination, samples of

frozen leaves (10�4 kg) were powdered in liquid N2 and

homogenized in a volume of 10�6 m3 containing 0.36 mol m�3

trichloroacetic acid (TCA) for 3 min. The homogenate was

centrifuged at 12,000 g for 15 min at 4 �C, and 5 � 10�7 m3

aliquots of the supernatant were mixed with 2 � 10�6 m3 of

1.22 mol m�3 TCA containing 34.54 � 10�3 mol m�3 thio-

barbituric acid (TBA). The mixture was heated at 95 �C for

30min in hermetically closed tubes and then cooled quickly in

an ice bath. The absorbance was read at 532 nm, and the

readingswere corrected for unspecific turbidity by subtracting

the absorbance at 660 nm. The TBARS concentration was

calculated using the molar extinction coefficient of

10�3 � 155 mol m�3 cm�1. The results were expressed in

10�3 mol (kg FW)�1.

2.7. Enzyme extraction and activity assays

Samples of frozen leaves (10�4 kg) were powdered in liquid

nitrogen and extracted with 0.1 mol m�3 TriseHCl buffer (pH

8.0) containing 10�2 mol m�3 DTT, glycerol 20% (v v�1) and

30 kg m�3 PEG-6000 [29]. For superoxide dismutase (SOD) and

ascorbate peroxidase (APX) extraction, the pH of the buffer

was adjusted to 7.0 and the 10�3 mol m�3 ascorbate was

added. The crude extract was centrifuged at 14,000 g for

30min at 4 �C, and the supernatant was used as the enzymatic

extract.

The activity of superoxide dismutase (SOD; EC: 1.15.1.1)

was determined by adding leaf extract to amixture containing

5 � 10�2 mol m�3 potassium phosphate buffer (pH 7.8),

10�4 � mol m�3 EDTA, 13 � 10�3 mol m�3 L-methionine,

2 � 10�6 mol m�3 riboflavin, and 75 � 10�6 mol m�3 p-nitro

blue tetrazolium chloride (NBT) in the dark. The reaction was

carried out under illumination (30Wfluorescent lamp) at 25 �Cfor 6 min. The absorbance was measured at 540 nm of wave-

length in spectrophotometer (Thermo Genesys 5, USA) [30].

One SOD activity unit (AU) was defined as the amount of

enzyme required to inhibit 50% of the NBT photoreduction,

and the activity was expressed as AU (kg FW min)�1.

The activity of ascorbate peroxidase (APX; EC: 1.11.1.1) was

assayed after reaction of the extract in the presence of

5 � 10�2 mol m�3 potassium phosphate buffer (pH 6.0) and

5 � 10�3 mol m�3 ascorbic acid. The reaction was started by

adding 10�7 m3 of 30 � 10�6 mol m�3 H2O2, and the decreasing

absorbance at 290 nm was monitored for 5 min [31]. APX

activity was calculated using the molar extinction coefficient

of the ascorbate (28 � 10�3 mol m�3 cm�1) and expressed as

10�3 mol ASA (kg FW min)�1.

The activity of catalase (CAT; EC: 1.11.1.6) was determined

after reaction of the enzymatic extract in the presence of

5 � 10�2 mol m�3 potassium phosphate buffer (pH 7.0) con-

taining 2 � 10�2 mol m�3 H2O2. The reaction took place at

30 �C, and the absorbance at 240 nm was monitored for 5 min

[32]. CAT activity was calculated according to the molar

extinction coefficient of H2O2 (36 � 10�2 mol m�3 cm�1) and

expressed as 10�3 mol (kg FW min)�1.

2.8. Data correlational analysis

The experiment was arranged in a completely randomized

design with five treatments and four independent replicates,

each consisting of an individual pot containing a single plant.

Data were analyzed by ANOVA, and mean values were

compared by the Tukey test at a confidence level of 0.05.

Linear regressions were performed to evaluate the relation-

ships between the most important variables obtained from

absolute data from plants exposed to different water deficit

levels.

3. Results

The responses of leaf water relations to drought treatments

indicate that J. curcas is a drought tolerant plant species. The

leaf water potential (Jw) gradually declined with decreasing

water supply; however, it reached relatively high values in the

most drought-stressed leaves (�1.05 MPa). In addition, the

hydration status of leaves did not change as indicated by the

relative water content, which was similar to well-watered

plants, as previously reported [78]. Stressed leaves did not

exhibit any visual symptoms of drought-induced injuries such

as drying, necrotic or chlorotic areas indicating that J. curcas

plants were able to restrict severe damages when exposed to

drastic water deficit conditions.

3.1. Gas exchange, chlorophyll-fluorescence, leaf dryweight, chlorophyll content and sugar accumulation

After ten days of exposure to water deficit, the relative values

(% of control) of leaf CO2 assimilation rate (PN), stomatal

conductance ( gS) and PN/CI ratio decreased significantly as the

water deficit increased (Fig. 1AeB) while the ETRs’/PN ratio

were significantly increased (Fig. 1B). The leaf DW and chlo-

rophyll content progressively decreased as the water deficit

increased (Fig. 1C). At the substratewatermass fraction of 10%

(the most severe drought stress), the leaf DW and chlorophyll

contentwere reduced by 35% and 32%, respectively, compared

to control (well-watered) plants.

The actual quantum yield of PSII (DF0/F0M) decreased grad-

ually as the water deficit increased but the maximal quantum

yield of PSII (FV/FM) did not differ significantly between

stressed and non-stressed plants (Fig. 2A). The apparent

electron transport rate (ETRs’) decreased significantly in all

water-deficit treatments. Inversely, the relative excess of light

energy (EXC) prominently increased as the water deficit rose

(Fig. 2B). The minimum chlorophyll fluorescence (FO)

increased slightly as water deficit increased (Fig. 2C) and the

non-photochemical quenching (NPQ) greatly increased

compared to the control plants. For instance, NPQ values were

around 2.6 times higher in plants subjected to themore severe

water deficit compared to well-watered control (Fig. 2C).

The leaf concentration of reducing sugars and sucrose

were strongly enhanced with decrease of water availability.

Total non-structural carbohydrate concentration significantly

Fig. 1 e Relative variation in CO2-assimilation rate (PN) and

stomatal conductance ( gS) (in A), instantaneous

carboxylation efficiency (PN/CI) and the ratio between the

apparent electron transport rate and CO2 assimilation

(ETRs’/PN) (in B) and leaf dry weight (DW) and chlorophyll

(Chl) concentration (in C) in J. curcas plants exposed to

water-deficit treatments for ten days. The absolute values

of the control (100%) were PN [ 9.98 mmol mL2 sL1;

gS [ 0.30 mol mL2 sL1; PN/CI [ 0.28 mmol mL2 sL1 PaL1

and ETRs’/PN [ 21.3 mmol mmolL1; Leaf

DW [ 0.141 kg plantL1; Chl concentration [ 0.88 g kgL1

FW. Data refer to mean values (n [ 4). Symbols with the

same letters are not significantly different by the Tukey

test ( p < 0.05).

Fig. 2 e Relative variation in maximal (FV/FM) and actual

(DF0/F0M) quantum yield of PSII (in A), apparent electron

transport rate (ETRs’) and relative energy excess (EXC) (in B)

and minimum fluorescence (FO) and non-photochemical

quenching (NPQ) (in C) in J. curcas plants exposed to water-

deficit treatments for ten days. The absolute values of the

control (100%) were DF0/F0M [ 0.663; FV/FM [ 0.774;

ETRs’ [ 106.3 mmol mL2 sL1; EXC [ 0.18; FO [ 482 and

NPQ [ 0.247. Data refer to mean values (n [ 4). Symbols

with the same letters are not significantly different by the

Tukey test ( p < 0.05).

b i om a s s an d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 7 0e2 7 9274

increased only in the two most severe levels of water deficit

(Fig. 3AeB). The sucrose concentration increased significantly

due to water deficit, reaching values 200% higher in the most

water-stressed plants as compared to control plants. On the

other hand, the starch concentration decreased gradually

with increasing water deficit, showing an inverse pattern of

response when compared to changes in sucrose and non-

structural sugar (Fig. 3AeB).

3.2. Oxidative damage and activities of SOD, APX andCAT

The electrolyte leakage (EL) and lipid peroxidation (given by

TBARS accumulation), indexes of membrane damage, were

markedly enhanced by stressful conditions. In the 10% water-

deficit treatment, EL and TBARS concentration were approxi-

mately 110% higher than in control plants (Fig. 4A). Similarly,

leaf H2O2 concentration increased substantially in all stressed

Fig. 3 e Relative variation in total non-structural

carbohydrates and reducing sugar concentrations (in A)

and sucrose and starch concentration (in B) in leaves of J.

curcas plants exposed to water-deficit treatments for ten

days. The absolute values of the control (100%) were total

non-structural carbohydrate

concentration [ 655.2 3 10L3 mol (kg DW)L1; reducing

sugar concentration [ 135.1 3 10L3 mol (kg DW)L1;

sucrose concentration [ 35.9 3 10L3 mol (kg DW)L1 and

starch concentration [ 484.2 3 10L3 mol (kg DW)L1. Data

refer to mean values (n [ 4). Symbols with the same letters

are not significantly different by the Tukey test ( p < 0.05).

Fig. 4 e Relative variation in electrolyte leakage (EL) and

lipid peroxidation (in A), H2O2 content and SOD activity (in

B) and APX and CAT activities (in C) in leaves of J. curcas

plants exposed to water-deficit treatments for ten days.

The absolute values of the control (100%) were EL [ 16%;

lipid peroxidation [ 39 3 10L3 mol TBARS (kg FW)L1;

H2O2 [ 5.86 3 10L3 mol (kg FW)L1; SOD [ 2.52 UA

(kg FW min)L1; APX [ 11.17 3 10L3 mol AsA

(kg FW min)L1 and CAT [ 33 3 10L3 mol H2O2

(kg FW min)L1. Data refer to mean values (n [ 4). Symbols

with the same letters are not significantly different by the

Tukey test ( p < 0.05).

b i om a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 7 0e2 7 9 275

plants compared to non-stressed ones. In plants subjected to

water deficit, the leaf H2O2 concentration increased by about

45% compared to control conditions (Fig. 4B).

The activities of the antioxidant enzymes catalase (CAT),

ascorbate peroxidase (APX) and superoxide dismutase (SOD)

presented differential response pattern to water deficit

conditions. The SOD activity increased slightly (w20%) in all

water-deficit treatments compared to control conditions

(Fig. 4B). APX activity was notably stimulated by water deficit,

with the most stressed plants reaching values 100% higher

than control plants (Fig. 4C). Conversely, CAT activity was

strongly inhibited in plants under water stress. In the two

most severe water-deficit treatments, CAT activity decreased

by 60% compared to the control condition (Fig. 4C).

3.3. Data correlational analysis

In order to assess an integrative analysis involving leaf CO2

assimilation, carbohydrate metabolism, photochemical

activity and oxidative protection mechanisms in drought-

stressed plants we performed a data correlational statistical

analysis. The concentrations of sucrose and reducing sugars

were inversely and significantly correlated with the leaf CO2

assimilation in J. curcas leaves (Table 1). Sucrose concentration

was themost closely and inversely correlated (r ¼ 0.9583) with

photosynthesis, whereas the starch concentration was

directly correlated (r ¼ 0.9871).

As expected, CO2 assimilation was positively correlated

with ETRs’ (r ¼ 0.9736) and inversely correlated with both EXC

Table 1 e Correlation analysis between sugars concentrations, CO2 assimilation rate, photochemical parameters andantioxidative enzymatic activities in leaves of J. curcas plants exposed to water-deficit treatments for ten days.

Correlations Equation Regression coefficient (r) Significance

Sucrose � PN y ¼ �20.546x þ 130.54 0.9583 p < 0.05

Starch � PN y ¼ 38.094 þ 289.28 0.9871 p < 0.05

Total soluble sugar � PN y ¼ �99.697x þ 612.25 0.9019 p < 0.05

ETRs’ � PN y ¼ 13.791x þ 41.923 0.9736 p < 0.05

EXC � PN y ¼ �0.1x þ 0.6459 0.9630 p < 0.05

NPQ � PN y ¼ �0.5486 þ 2.7628 0.9728 p < 0.05

EXC � ETRs’/PN y ¼ 0.0096x þ 0.0197 0.9722 p < 0.05

EXC � NPQ y ¼ 0.1822x þ 0.1425 0.9896 p < 0.05

EXC � APX y ¼ 0.2725x�0.1227 0.9407 p < 0.05

EXC � CAT y ¼ �0.0205xþ0.8077 0.8861 p < 0.05

CAT � ETRs’/PN y ¼ �1.852x þ 75.911 0.7936 p < 0.05

CAT � H2O2 y ¼ �5.8633x þ 66.886 0.9269 p < 0.05

H2O2 � EL y ¼ 8.2115x�26.907 0.9922 p < 0.05

H2O2 � TBARS y ¼ 5.5134x�15.752 0.6892 p < 0.05

b i om a s s an d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 7 0e2 7 9276

andNPQ (data not shown). Such correlations indicate that part

of the excess energy was dissipated as heat in drought-

stressed J. curcas leaves. The EXC was also positively corre-

lated with the ETRs’/PN ratio (r ¼ 0.9722), indicating that the

excess of electrons potentially available to NADPþ reduction

and further utilization in biochemical CO2 fixation were

transferred to photorespiration.

APX activity was positively correlated with EXC (r ¼ 0.9407)

while CAT activity was inversely correlated with EXC

(r ¼ 0.8861). These results indicate that APX and CAT activities

were inverselymodulated by energy excess at the PSII level. In

addition, CAT activity was inversely correlated with both

ETRs’/PN (r ¼ 0.7936) and leaf H2O2 concentrations (r ¼ 0.9269).

These data indicate that H2O2 accumulation in stressed leaves

might be correlated with both increased photorespiration and

decreased CAT activity. Interestingly, the leaf H2O2 concen-

trations were more closely correlated with electrolyte leakage

(r ¼ 0.9922) than with lipid peroxidation (r ¼ 0.6892) (Table 1).

4. Discussion

4.1. J. curcas leaves display coordinate mechanismsinvolving CO2 assimilation and photochemical activity toalleviate photodamage under drought conditions

We have previously shown that water deficit induces

decreases in CO2 assimilation in J. curcas leaves by stomatal

and biochemical limitations [22,33]. Herein, we observed that

PN/CI ratio (photosynthetic carboxylation efficiency) progres-

sively decreased as the water deficit increased e reinforcing

that CO2 assimilation was limited by biochemical limitations.

This impairment in biochemistry of photosynthesis was

inversely correlated with increasing in reducing sugars and

sucrose concentrations. In such situation, it might be argued

that sugars can have played an important regulatory role in

the CO2 assimilation of drought-stressed J. curcas plants, likely

through a negative feedback mechanism. In fact, the regula-

tion of photosynthesis by sugars through source-sink rela-

tionship has been widely reported in plants under water

deficit [9].

Among the possible mechanisms of photosynthesis

modulation by sugar accumulation might be speculated

a decrease in re-cycling of Pi from cytosol to chloroplasts

associatewith decreases in ATP andNADPH consumption and

reduced regeneration of RuBP in Calvin cycle [34]. Moreover,

decrease in expression of photosynthesis-related genes [19],

shifting in cycling of sucrose and hexoses [35], damage to

thylakoid structure and chlorophyll degradation under high

carbohydrate accumulation might also contribute to photo-

synthesis modulation [18].

The sucrose and reducing sugar concentrations were

inversely correlated with starch concentration in J. curcas

leaves suggesting the occurrence of a drought-modulation of

starch hydrolysis associated with lower sucrose translocation

from leaves to sink tissues due to growth restriction as

demonstrated in cucumber plants [9]. As found here, increase

in the soluble sugar fraction was accompanied by a sharp

decrease in the starch concentration in plants experiencing

low leaf water potential [19]. Couee et al. [15] have reported

that such a pattern of carbohydrate degradation and synthesis

plays an important role in the accumulation of soluble sugars

in cells. The accumulation of soluble sugars suggests a shift in

carbon metabolism as a consequence of water deficit.

McCormick et al. [35] have reported that the accumulation of

soluble sugars is a result of metabolic impairment that affects

either the composition and translocation of sugars in leaves.

They also suggested that this responsemight contribute to the

inhibition of photosynthesis during water stress.

Interestingly, in this study the drought-induced biochem-

ical restrictions on photosynthesis were inversely correlated

with ETRs’/PN ratio (an indicator of alternative electron sink)

indicating that drought might had induced progressive

increase in photorespiration [20,22]. Increases in photorespi-

ration have been considered to be a protective mechanism,

consuming photochemical products and impeding damage at

the PSII level [5]. Although decreases in photochemical

activity have been observed in J. curcas plants under water

deficit, we argue that leaf tissues would be more affected if

alternative electron sinks, such as photorespiration, were

non-active. In fact, the maximal quantum yield of PSII (FV/FM)

was not affected by water deficit (Fig. 2), indicating no

b i om a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 7 0e2 7 9 277

photodamage to PSII reaction centers even with plants

showing reduced chlorophyll content.

The reduction in chlorophyll concentration of J. curcas

leaves subjected to water deficit may be an additional strategy

of photo-protection reducing light absorption and thus

decreasing energetic pressure at the PSII level [5]. In parallel to

biochemical restrictions (PN/CI) caused by water deficit, the

actual quantum yield of PSII and the apparent electron

transport rate were progressively decreased by water deficit

(Fig. 3). As ETRs’ is considered an overall index of photo-

chemical activity, our data suggest that the water-deficit

treatments imposed significant de-activation of the electron

transport chain in the thylakoid membranes [25,6]. This

phenomenon is probably associated with damage caused on

the primary electron acceptors of PSII (plastoquinone) due to

accumulation of the reduced Qa [9] as indicated by the

significant reduction in DF0/F0M. We also noticed strong

increases in EXC and slight increases in FO. High EXC under

water-deficit conditions indicates an over-excitation of the

PSII complex by high available light energy, which must be

deactivated by dissipating processes to avoid photochemical

damage. The slight effect on FO suggests that thylakoid

membrane structure and partial dissociation of LHCII from

PSII were relatively preserved [25].

Even in plants showing high EXC and reduced ETRs’, the

water deficit did not cause photoinhibition of photosynthesis

in J. curcas, as indicated by the lack of significant alteration in

FV/FM. In addition to reduced light absorption due to decreases

in chlorophyll concentration and increased alternative elec-

tron sinks (high ETRs’/PN), plants also showed an increase in

non-photochemical quenching (NPQ), indicating the occur-

rence of a non-radiative energy dissipation mechanism by

thermal processes [20]. This photoprotective mechanism

helps to maintain the high oxidative state of the primary

electron acceptors of PSII, reducing the probability of photo-

damage and photo-oxidative stress in chloroplasts [25].

4.2. The role of SOD, APX, and CAT activities and sugarsin photo-oxidative protection

The correlational analysis performed in this study strongly

suggests that sucrose and reducing sugars might have nega-

tively modulated the CO2 assimilation in drought-stressed J.

curcas leaves. In addition, sugars might play a central role in

photochemical apparatus protection and ROS scavenging in

stressed plants [15]. The relationship between sugars and ROS

production or between sugars and ROS responses is not

a straightforward positive correlation; high sugar levels can

correspond to the activation of some ROS-producing path-

ways and the de-activation of other ROS-producing pathways,

and both high and low sugar levels can result in enhanced ROS

responses [15]. Sugars, especially glucose and sucrose, seem to

play a dual role with respect to ROS, either promoting ROS

production or participating indirectly in ROS scavenging

mechanisms through the processes of NADPH-generating,

such as the oxidative pentose-phosphate pathway [36].

Sugars are also involved in regulating the expression of

several photosynthesis-related genes as well as some ROS-

related genes, such as superoxide dismutase [15]. Therefore,

it has been suggested that sugars may function as plant

signals that are useful in sensing and controlling not only

photosynthetic activity but also the cellular redox balance

[15]. In this context, our results suggest that sugars might act

as protective agents and may be also involved effectively in

the osmotic adjustment of J. curcas plants [21].

Photochemical reactions of photosynthesismay lead to the

accumulation of ROS via the Mehler reaction and other

processes such as energy excess in photosystems. In fact, the

energy excess in the thylakoids allows the formation of

superoxide radicals by the waterewater cycle. The first

defense line against accumulation of these toxic radicals is the

SOD isoforms localized in chloroplasts [16]. In this study SOD

activity was slightly stimulated in drought-stressed plants

perhaps through a drought-induced up-regulation of gene

expression and/or activation of protein isoforms. SOD scav-

enges the O$�2 generated by the electron transport chain in the

chloroplasts and the H2O2 produced by SOD activity is then

eliminated by APX [17]. Increases in SOD activity in leaves of

drought-stressed plants had been widely reported [37]. This

response in J. curcas leaves may indicate that its chloroplasts

have an effective mechanism of superoxide scavenging.

APX activity increased strongly in J. curcas leaves as the

water deficit increased, indicating a provable efficient mech-

anism against H2O2 accumulation in chloroplasts. In addition,

the APX activity was positively correlated with energy excess

at the PSII level, suggesting that drought-induced modulation

in the expression and/activity of chloroplast APX isoforms

could have occurred. Several APX isoforms are widely

distributed in almost all cell organelles, and abiotic stresses

frequently induce increases in both the gene expression and

activity of APX to compensate for deficiencies in CAT activity

[38]. Our data reveal that the increase in total APX activity

partially compensated for the restriction presented by CAT

activity because the accumulated H2O2 in leaf tissue did not

increase in the most severe water-deficit treatment.

In contrast to SOD and APX, CAT activity was strongly

inhibited underwater deficits (Fig. 4). This inhibitionmay have

been a consequence of down-regulation of gene expression

and/or degradation, denaturation or inhibition/inactivation of

this protein [10]. CAT removes the H2O2 generated in the

photorespiration pathway inside the peroxisomes [39]. Under

drought stress thephotorespiratory activity is commonly over-

induced and as a consequence large amounts of hydrogen

peroxide are produced in peroxisomes [40]. The excess H2O2

produced in peroxisomes might diffuse to the cytosol and be

converted to hydroxyl radicals by the Fenton reaction [41].

These are themost toxic ROS and they are directly involved in

lipid peroxidation at plasmalemma [17]. In this study, CAT

activity was inversely correlated with hydrogen peroxide

accumulation and with ETRs’/PN, two photorespiration indi-

cators. These data suggest that CAT activity in J. curcas leaves

was not able to sustain the drought-induced photorespiration

and scavenging H2O2 excess.

The oxidative damages caused by H2O2 were indicated by

increases in lipid peroxidation and electrolyte leakage in leaf

tissues. In fact, several studies have shown that lipid perox-

idation is a natural metabolic process under normal aerobic

conditions and it is one of the most investigated ROS actions

[17]. It is reported that ROS bring peroxidation of membrane

lipids leading to oxidative damage [6]. However, despite the

b i om a s s an d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 7 0e2 7 9278

drought stress has induced strong lipid peroxidation in J.

curcas leaves, to date, have no any experimental evidences to

indicate the extension of oxidative damages on the physio-

logical performance of stressed plants [17].

In general, our data strongly suggest that J. curcas leaves

display an effective mechanism against drought-induced

photo-oxidative damages. The first defense line utilizes a coor-

dinate balance involving reduction in light absorption and

dissipation of energy excess at the PSII by non-photochemical

quenching. Sucrose and reducing sugars appear to play

a central role in the regulation of photosynthesis. These sugars

could contribute to an adequate balance between the rates of

CO2 assimilation and the supply of electrons by photosynthetic

electron transport chain and contributing to oxidative protec-

tion. The photorespiratory activity and NPQ are important

processes for dissipation of the excessive energy. The oxidative

damage protection in the chloroplasts involves an effective

participation of SOD and APX activities restricting the accumu-

lationexcessiveofsuperoxideandhydrogenperoxide.Theother

defense line is representedby theCATactivitywhich isessential

to scavenging H2O2 excess produced by photorespiratory cycle.

This enzymewas apparently less effective in J. curcas subjected

todroughtstressbecause inwater-stressed leaves theactivityof

this enzyme is strongly inhibited.

5. Conclusion

In conclusion, our data suggest that drought stress induces

a coordinate response on the photosynthesis (photochemistry

and carboxylation phases), which could be modulated by

sugar accumulation. The antioxidant enzymatic protection

represented by SOD and APX activities was beneficial for

oxidative damage protection. Moreover, the accumulation of

sugars would have contributed for cellular protection and

partial preservation of key physiological processes in drought-

stressed plants. However, the drought-induced decrease in

the CAT activity could be a limiting factor for H2O2-scavenging

and oxidative protection in J. curcas under severe water deficit

conditions.

Acknowledgments

We thank the Fundacao Cearense de Apoio ao Desenvolvi-

mento Cientıfico e Tecnologico e FUNCAP (Project 2155/PRO-

NEX) and the Conselho Nacional de Desenvolvimento

Cientıfico e Tecnologico (CNPq) for financial support. The

authors would like to thank the Fazenda Tamandua for

supplying of the Jatropha curcas seeds.

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