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easonal variations of photosynthesis, gas exchange, quantum efficiency ofhotosystem II and biochemical responses of Jatropha curcas L. grown inemi-humid and semi-arid areas subject to water stress
laudiana Moura dos Santos, Valtair Verissimo, Humberto Cristiano de Lins Wanderley Filho,ilma Marques Ferreira, Polyana Geysa da Silva Cavalcante, Eduardo Vicente Rolim, Laurício Endres ∗
aboratorio de Fisiologia Vegetal, Centro de Ciencias Agrarias, Universidade Federal de Alagoas, Av. Lourival Melo Mota, s/n, Cidade Universitária, Maceió, AL, CEP 57072-900, Brazil
r t i c l e i n f o
rticle history:eceived 30 January 2012eceived in revised form 29 March 2012ccepted 5 April 2012
eywords:as exchangehlorophyll a fluorescencexidative stresssmotic adjustment
a b s t r a c t
This study aimed to compare the physiological adaptations of Jatropha curcas in two regions with differentclimates, the Agreste (a semi-arid tropical climate) and the Florest zone (a semi-humid tropical climate)subject to water stress. Measurements of gas exchange and photochemical and biochemical efficiencywere performed in two seasons, the dry season (September to February) and the rainy season (Apriland August), between 2006 and 2007. During the dry season in the Florest zone, the J. curcas plantspresented better photosynthetic performance, with smaller decreases in the rates of photosynthesis,stomatal conductance and transpiration because the conditions of water availability in the soil wereslightly higher than in the semi-arid region. A greater vapor pressure deficit was observed during the dryseason of the semi-arid region, showing a negative correlation with the stomatal opening of the leaves,which contributed to a reduction in the values of the net photosynthetic rate. During the dry season in
the semi-arid region, dynamic photoinhibition occurred in the leaves of J. curcas around mid-day, witha rapid recovery during the afternoon. However, chronic photoinhibition may have occurred in someplants with a severe water deficit. A reduction in the effective quantum efficiency was also observedduring the driest months. The increase of catalase activity and the accumulation of proline, total solublesugars and amino acids during periods of low water availability in the soil suggest that J. curcas presentsan efficient protection mechanism that allows the plant to survive under drought conditions.
. Introduction
Drought is an abiotic factor that limits the growth and pro-uctivity of crops worldwide and can result in the significanteduction of photosynthetic rates, resulting in lower growth ratesnd productivity and causing serious socio-economic and envi-onmental losses (Johari-Pireivatlou et al., 2010). Furthermore, the
se of drought-tolerant crops has been proposed for the economicxploitation of non-cultivated and marginal areas (Behera et al.,010).
Abbreviations: A, photosynthetic rate; A/E, instantaneous water use efficiency;/Ci , instantaneous carboxilation efficiency; A/gs, intrinsic water use efficiency; AET,ctual evapotranspiration; CAT, catalase; Ci , internal concentration of CO2; E, tran-piration rate; FAA, free amino acid; Fv/Fm, maximum quantum yield of photosystemI; gs, stomatal conductance; Pro, proline; ROS, reactive oxygen species; RH, relativeir humidity; TSS, total soluble sugars; Tl , leaf temperature; Tc, camera temperature;PDleaf-to-air, leaf-to-air vapor pressure deficit; ˚PSII , effective quantum yield of thehotosystem II.∗ Corresponding author. Tel.: +55 82 8831 3055; fax: +55 82 3261 1351.
Jatropha curcas L. is a large perennial shrub that belongs to theEuphorbiaceae family; the plant grows rapidly, reaching a height of3–5 m, but it can reach a height of 8 m under favorable conditions(Divakara et al., 2010). The species has great economic potential andis used in the pharmaceutical and cosmetic industries, yet greatereconomic interest lies in the seeds, which are used in the productionof oils for biodiesel (Kheira and Atta, 2009). J. curcas is found fromthe northeast to southern regions of Brazil (Saturnino et al., 2005),growing under very different climatic characteristics from the restof the country. The semi-arid Northeastern region is characterizedby a negative hydric balance, with a mean annual rainfall of lessthan 800 mm, a mean insolation of 2800 h year−1 and mean annualtemperatures of 23–27 ◦C. These conditions lead to high evapo-transpiration rates, setting up a water deficit in nearly the entireregion (Sudene, 2010); thus, fewer profitable agricultural optionsare available to sustain farmers in rural areas. Therefore, J. curcasmay be an interesting agricultural option for this region.
J. curcas has gained prominence as a native species that isstrongly tolerance to a water deficit (Maes et al., 2009a; Pompelliet al., 2010), and it is beginning to have economic importance inthe world, as it is grown in the tropical and subtropical regions of
everal countries in Africa, Asia and Latin America. Furthermore, its grown mainly in arid and semi-arid regions, which exhibit a highvaporative demand and low water availability (Meng et al., 2009;aes et al., 2009b). J. curcas can be cultivated under various cli-atic conditions, with rainfall between 200 and 1500 mm year−1,
ut it grows best in locations with an annual rainfall above 600 mmKheira and Atta, 2009).
Extreme environmental conditions, such as high temperature orrought, can affect the photosynthetic activity in plants (Yu et al.,009). Photosynthesis is particularly sensitive to a water deficitecause the stomata close to conserve water as the soil water statusecreases (Lawlor and Tezara, 2009) indeed, stomatal regulation isital in maintaining the hydric status of the plant (Guo et al., 2010).
decrease in the net photosynthesis rate and transpiration is usu-lly observed in plants under drought conditions (Endres, 2007;essini et al., 2009; Saibo et al., 2009) and is also observed in envi-
onments where the evaporative demand is very high (Feng andao, 2005). As a result, photochemical damage to photosystem IIan be induced under such conditions (Zheng et al., 2009; Saibot al., 2009; Silva et al., 2010a). Furthermore, the over-excitation ofhe reaction centers of photosystem II may increase the productionf reactive oxygen species (ROS) in chloroplasts (Carvalho, 2008). Inlant cells, antioxidant enzymes, including superoxide dismutaseSOD) and catalase (CAT), are considered members of the oxidativeefense system, functioning in the protection of cells against thedverse effects of ROS (Scandalios, 2005; Noctor and Foyer, 2005;sada, 2006; Gao et al., 2008).
The accumulation of metabolites may also be important for plantolerance to stressful environments. The accumulation of solutes,uch as proline, which acts as a mediator of osmotic adjustmenty protecting the integrity of the plasma membrane, a sourcef carbon and nitrogen (Claussen, 2005; Cordeiro et al., 2009;ohari-Pireivatlou et al., 2010) and an ROS scavenger (Valliyodannd Nguyen, 2006), is a significant metabolic change under aater deficit. In addition, studies have demonstrated the accu-ulation of soluble sugars, glycine betaine, soluble amino acids
nd soluble protein (Yousifi et al., 2010; Babita et al., 2010; Silvat al., 2010a; Pompelli et al., 2010) in plants under stress. Sucholutes may accumulate to high levels under conditions of a loweaf water potential, thus protecting cells against dehydrationhrough osmotic adjustment (Cordeiro et al., 2009; Zhou and Yu,010).
The objective of the study presented here was to evalu-te the adaptation of J. curcas to two environmental conditions,emi-humid and semi-arid, which are subject to water stress, inortheastern Brazil.
. Materials and methods
.1. Field study description
The studies of J. curcas were conducted in two distinct regionsf the Alagoas State. One region is located in the Rio Largoity, in the Florest zone, a region of the Coastal Plains (09◦27′Snd 35◦49′W, mean altitude of 127 m). The region is charac-erized by a semi-humid tropical climate with a rainy seasonetween April and August and a dry season from September toebruary (spring–summer), with mean rainfall of approximately818 mm year−1, with the wettest month in June (294 mm) andhe driest in December (41 mm) (Souza et al., 2005; CPTEC, 2011).his region is located in the tropical forest of the Atlantic For-
st, which is considered the second richest biome and one of theost endangered in the world (Myers et al., 2000), presenting a
ubperennial forest type of vegetation, with portions of evergreenorest (Resende et al., 2002).
and Products 41 (2013) 203– 213
The second study area was the Igaci city, in the Agreste (09◦33′Sand 36◦38′W, altitude 240 m), which has a type of semi-aridtropical climate, with a well-defined rainy season between themonths of April to August and a dry period during the sum-mer months. This region has a mean rainfall of 740.51 mm, withthe wettest month in May (127.3 mm) and the least rainy inNovember (11.2 mm) (Agritempo, 2006). This region is located ina transition area between the hinterland and the Atlantic For-est, and the vegetation is basically composed of hyperxerophilousCaatinga, with stretches of Deciduous Forest (Mascarenhas et al.,2005).
The temperature and rainfall during the experimental periodwere obtained from an automatic weather station installed in aopen grassed area about 500 m from the experimental area inthe Forest Zone and from weather station in DNOS Arapiracaabout 15 km from the experimental area in the Agreste. The soilwater store and field capacity and actual evapotranspiration wereobtained from CPTEC (CPTEC, 2011).
Four hundred plants, originated from seeds, were planted ineach region and cultivated in 3 × 2 spacing. The plants were eval-uated between 7 and 21 months after sowing, between the periodof October 2006 to December 2007. The physiological tests werecarried out at five different times, always on similar dates in thetwo studied regions. The evaluations were made on 10/13/2006,12/11/2006, 07/25/2007, 10/03/2007 and 12/18/2007 in thesemi-humid region and on 11/02/2006, 12/15/2006, 07/03/2007,09/25/2007 and 12/19/2007 in the semi-arid region.
2.2. Gas exchange measurements
Gas exchange measurements were performed only during thedry season. On each date, a daily curve of gas exchange wasconstructed using at least four time points throughout the day,between 6:00 a.m. and 4:00 p.m., with intervals of approximately2 h.
Fully expanded leaves of the third pair from the apex of tendifferent plants were marked, and the gas exchange measurementswere taken every 2 h (always in the same leaf) throughout the day.
The gas exchange measurements were performed with aportable infrared CO2 analyzer (IRGA, ADC Bioscientific Ltd., Hod-desdon, UK), with a light source of 1123 mol m−2 s−1. The followingvariables were evaluated: rate of photosynthesis (A), transpira-tion (E), stomatal conductance (gs), intracellular CO2 concentration(Ci), leaf temperature (Tl), chamber temperature (Tc) and relativehumidity in the chamber (RH). The CO2 concentration inside thechamber, air humidity and temperature varied depending on theenvironmental conditions. The ratio of instantaneous water useefficiency (A/E) and the intrinsic water use efficiency (A/gs) werecalculated using the measured values of A, E and gs.
The vapor pressure deficit between the leaf and the air,VPD(leaf-air), throughout the day was obtained by calculating thedifference between the saturation (es) and real (e) air pressuresaccording to the method of FAO (1991), using measurements of theleaf temperature and relative humidity in the chamber as follows:VPD(leaf-air) = (es − e) in kPa.
VPD = es − e =(
0.6108 ∗ exp[
17.27 ∗ Tf
273.3 + Tf
])−
(es − UR
100
)
2.3. Chlorophyll a fluorescence analysis
The photochemical efficiency of photosynthesis was obtained
through the evaluation of the chlorophyll a fluorescence on thesame leaves for which the gas exchange was evaluated. The mea-surements of the maximum quantum efficiency of PSII (Fv/Fm) weredetermined after the leaves were adapted to the dark for 20 min,
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C.M. dos Santos et al. / Industrial
sing a modulated fluorometer 051-FL (OPTI-SCIENCES, Hudson,H, USA). The measurements were taken after the exposure to
aturating light pulses of 1 s, to promote the closure of the PSIIeaction centers, according to the method described by Maxwellnd Johnson (2000), two measurements were taken for each leafrom ten different plants with a total of 20 repetitions in each timeoint.
The effective quantum efficiency was measured at 12:00 p.m.ccording to Baker and Rosenqvist (2004). There were performedhree measurements per leaf from ten different plants for a total of0 repetitions in each time point. The relative chlorophyll contentas estimated using a portable device (SPAD-502, Minolta Corpo-
ation, Ramsey, USA) on the same leaves as the fluorescence andas exchanges measurements with eight measurements per leaf,n ten different plants.
.4. Biochemical analyses
The organic solutes and enzyme activity were measured fromeaves collected from the same plants in which the gas exchange
as evaluated during the dry and rainy seasons, on 12/11/2006,7/25/2007, 10/03/2007 and 12/18/2007, only in the semi-humidropical region. The leaf samples were frozen in liquid nitrogen andtored at −20 ◦C until they were lyophilized and macerated for theetermination of the organic solutes.
The free proline in the lyophilized leaf tissue was measuredy a reaction with ninhydrin according to the method of Batest al. (1973), with modifications. Approximately 100 mg of tis-ue was macerated in liquid nitrogen and homogenized in 8 mLf 118 mM sulfosalicylic acid (5-sulfosalicylic acid 2-hidrate P.A.;7H6O6S·2H2O). The homogenate was then centrifuged at 3000 × gor 10 min at 25 ◦C, and the supernatant was used for the quantita-ive analysis.
The ninhydrin acid solution was prepared with 7 mmoles of nin-ydrin P.A. (C9H6O4) in 30 mL of glacial acetic acid (CH3COOH) and0 mL of 6 M phosphoric acid (H3PO4). In a screw-capped test tubes,
mL of extract was combined with 1 mL of ninhydrin acid and 1 mLf glacial acetic acid, and the mixture was incubated in a water bath100 ◦C) for 1 h. The extraction of the chromophore was performedy adding 2 mL of toluene. Known concentrations of proline weresed for the standard curve. The absorbance of the reaction mixtureas measured spectrophotometrically at a wavelength of 520 nm,
nd the results are expressed in �mol g−1 dry mass (DM).The concentration of soluble sugars was determined according
o a previously described colorimetric method (Dubois et al., 1956).n brief, 20 mg of lyophilized leaf sample was homogenized in 4 mLf distilled water and allowed to incubate for 1 h, followed by cen-rifugation at 3000 × g for 15 min at 25 ◦C. In a total volume of 7 mL,
mL of extract was combined with 1 mL 0.56 M phenol (C6H5OH)nd 5 mL of concentrated sulfuric acid. The carbohydrates wereuantified by measuring the absorbance at 490 nm, using d(+) glu-ose as a standard.
The measurements of the soluble amino-N were made using0 mg of lyophilized leaf tissue homogenized with 2 mL of 0.01 M-phosphate (pH 7.6) containing 0.1 M NaCl, with stirring every5 min for 1 h. The homogenate was then centrifuged at 3000 × gor 5 min at 4 ◦C. An aliquot of the supernatant (0.5 mL) was added to.5 mL 0.1 M trichloroacetic acid (Cl3CCOOH). After 1 h, the materialas centrifuged at 12,000 × g for 5 min at 4 ◦C, and the super-atant was processed as below. In screw-capped test tubes, 0.5 mLf extract, 0.25 mL 0.2 M sodium citrate buffer (pH 5) and 0.1 mL
80 mM ninhydrin (C9H6O4) were added to 17.7 M methyl Cel-
osolve (ethylene glycol P.A.; C2H6O2) and 0.5 mL 0.2 mM KCN in7.7 M methyl Cellosolve (ethylene glycol P.A.; C2H6O2). The mix-ure was incubated in a water bath at 100 ◦C for 20 min, and the
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absorbance was measured at 570 nm, using glycine as a standard(Yemm and Cocking, 1955).
2.5. Extraction and antioxidant enzyme activity
The activity of catalase (CAT) (EC 1.11.1.6) was determinedaccording to the methodology adopted by Havir and Mchale (1987),with modifications by Pompelli et al. (2010). An extract wasobtained from 30 mg of fresh leaf material by maceration in 2.7 mMPVPP (polyvinylpyrrolidone; [C6H9NO]n) and by adding 2 mL ofextraction buffer (50 mM K-phosphate buffer [pH 7.5], 2 mM EDTA,20 mM sodium ascorbate, 1.62 mM Triton X-100 [C34H62O11] anddeionized water). Once homogenized, the extract was centrifugedat 15,000 × g for 15 min at 4 ◦C; the supernatant was used for theanalysis.
The enzyme reaction (1 mL total) was performed in a 1-mL quartz cuvette containing 50 mM K-phosphate buffer (pH7.5), 12.5 mM H2O2 and 30 �L of enzyme extract; the volumewas brought to 1 mL with deionized water. The absorbance wasmeasured at 240 nm for 1 min, quantifying the decrease in the con-centration of H2O2. We adopted an extinction coefficient for H2O2of 36 mM−1 cm−1 at 240 nm for the calculation of the enzyme activ-ity. The values were expressed in CAT min−1 �g−1 of protein. Thesoluble protein was quantified by the Bradford method (1976).
2.6. Statistical analysis
The data were analyzed separately by analysis of variance andjointly by the Pearson correlation analysis (r) (Bussab and Morettin,1986). When suitable, means were compared by the Tukey test at5% probability.
3. Results
3.1. Climatological characterization
In both of the regions studied (semi-humid and semi-arid), twoseasons were observed, a dry and a rainy season, during the studyperiod of October 2006 to December 2007 (Fig. 1). The semi-humidregion exhibited a total rainfall of 1651.76 mm, with a maximum of230.63 mm in the month of August 2007 and minimum of 10.92 mmin the month of December 2006, and an average temperature of24.5 ◦C. As for the semi-arid region, the rainfall was 1154.5 mm,with a maximum of 187.6 mm in the month of May 2007 and a min-imum of 2.4 mm in the month of December 2006, and an averagetemperature of 25.1 ◦C (Fig. 1A and C). The average amplitude of theregion’s temperature was low, ranging from 22.1 ◦C to 26.6 ◦C in thesemi-humid and 22.35–27.65 ◦C in the semi-arid region (Fig. 1A).
During the months of the dry season in the semi-humid region,the highest real evapotranspiration (REE) was recorded in themonth of October 2006 (4.24 mm), and the lowest was recordedin the months of February 2006 (0.64 mm) and December 2007(1.15 mm). In the semi-arid region, the highest values were foundin the month of September 2006 (3.2 mm), and the lowest werefound in February 2006 (0.15 mm) and November 2007 (0.26 mm)(Fig. 1B). In general, the REE was always the highest in the semi-humid region due to the greater availability of water in theenvironment (Fig. 1C), as compared to the semi-arid region.
In the semi-arid region, the dry season was more prolongedand more pronounced than in the semi-humid region due to low
rainfall, and the water deficit of the soil began in the month ofSeptember 2006 and extended to February 2007. In the tropicalsemi-humid region, the water deficit of the soil was only found inthe months between November 2006 and February 2007 (Fig. 1C).
206 C.M. dos Santos et al. / Industrial Crops
Fig. 1. Climatic characteristics: (A) total monthly rainfall and mean tempera-tures, actual evapotranspiration (AET) (B), and soil water store (C) (horizontal barrepresents soil field capacity) in Agreste (semi-arid climate) and in Forest Zone(
S
3
tv(
5Vp
The instantaneous water use efficiency (A/E) had a strong neg-
semi-humid climate) of Brazil.
ource: CPTEC (2011).
.2. Diurnal variation of gas exchange
The differences in gas exchanges of J. curcas plants grown inhe semi-humid and the semi-arid regions (Fig. 2) reflected theariations in the climatic conditions of the local environmentFig. 1).
The VPD was higher during the dry period, with values up to
kPa and minimum of 1 kPa in the two regions (Fig. 2A and B). ThePD had a negative influence on the gas exchange of the J. curcaslants. When considering all of the gas exchange data during the
and Products 41 (2013) 203– 213
dry season, there was a negative correction between the VPD(leaf-air)and the stomatal conductance (R = −0.32**) and photosynthesis(R = −0.45**) (Table 1), though no influence on leaf transpiration(R = 0.02) was observed. In contrast, the more hydrated plants, witha stomatal conductance above 0.1 mol m−2 s−1, VPD showed a posi-tive correlation with leaf transpiration (r = 0.67**), causing a greatercooling of the leaf, which is suggested by the negative correlationbetween the VPD(leaf-air) and the Tf − Tc (R = −0.56**) (Table 1).
The stomatal conductance values ranged between 0.05 and0.6 mol m−2 s−1 in the plants of the semi-humid region (Fig. 2C)and between 0.01 and 0.35 mol m−2 s−1 in the plants of the semi-arid region (Fig. 2D). The lowest stomatal conductance values werefound in the afternoon between the hours of 12 p.m. to 14 p.m.,coinciding with the times of the higher VPD(leaf-air) (Fig. 2A and B).
The lowest level of stomatal conductance throughout the daywas found on 12/11/2006 (semi-humid region) and 12/15/2006(semi-arid region) (Fig. 2C and D, respectively), presenting valuesnear zero. This period was considered the driest in both locationswhen compared to the other times of the study (Fig. 1A and C), asindicated by a high VPD(leaf-air) (Fig. 2C and D) and low soil wateravailability (Fig. 1C).
In general, the leaf transpiration presented higher values inthe morning, with maximum values up to 8.0 mmol m−2 s−1 inthe semi-humid region (Fig. 2E) and up to 4 mmol m−2 s−1 in thesemi-arid region (Fig. 2F). In the semi-humid region, the leaf tran-spiration was found to be extremely low (throughout the day) onlyon 12/11/2006, whereas in the semi-arid region, the leaf transpira-tion was only higher than 1.0 mmol m−2 s−1 on 09/25/2007 (Fig. 2Eand F).
When considered all data from both sites, the leaf transpira-tion of the J. curcas plants presented a positive correlation withthe stomatal conductance (R = 0.35**), and it was not correlatedwith the VPD(leaf-air) (Table 1). While, when considered only thedata with the stomatal conductance higher than 0.1 mol m−2 s−1,a lower correlation was found between stomatal conductanceand transpiration (R = 0.17*), and a higher correlation betweenVPD(leaf-air) and transpiration appears (R = 0.67**). A decrease intranspiration also affected the leaf temperature, which was demon-strated by the negative correction between the two parameters(R = −49**).
Photosynthesis tended to decrease throughout the day at bothsites and followed a pattern similar to that of the stomatal con-ductance (Fig. 2A, B, G and H). The photosynthetic rates werehigher in the morning (at 8 a.m.) on 10/13/2006, 10/03/2007 and12/18/2007 in the semi-humid region, presenting values between12 and 20 �mol m−2 s−1 (Fig. 2G), followed by a reduction in thesevalues at mid-day of approximately 7 and 12 �mol m−2 s−1.
In general, photosynthesis was lower in the semi-arid regionon 11/02/2006, 09/25/2007 and 12/19/2007, with values between8 and 14 �mol m−2 s−1 at 8 a.m.; from noon, there was a reduc-tion in these values between 12 and 2 �mol m−2 s−1 (Fig. 2H).On 12/11/2006 (semi-humid region) and 12/15/2006 (semi-aridregion), the CO2 assimilation by the plant throughout the day wasvirtually non-existent due to the closing of the stomata during thisperiod.
The photosynthesis presented a positive correlation with thestomatal conductance (r = 0.32**) and a negative correlation withthe VPD(leaf-air) (r = −0.45**, Table 1). This correlation can clearly beobserved on 10/13/2007 and 18/12/2007 (semi-humid region) and09/25/2007 and 19/12/2007 (semi-arid region), when the photo-synthesis and stomatal conductance was high and VPD(leaf-air) waslow (Fig. 2).
ative correlation with the VPD(leaf-air) (R = −0.55**), perhaps due tothe influence of the VPD(leaf-air) on the transpiration (R = 0.67**)when the stomata had a conductance above 0.1 mol m−2 s−1
C.M. dos Santos et al. / Industrial Crops and Products 41 (2013) 203– 213 207
8
F8
E
0.0
0.2
0.3
0.5
0.6
6:00 8:00 10 :00 12 :00 14 :00 16 :00 18 :00
gs
(mo
l m
-2s-1
)
Time (hours)
D
0.0
0.2
0.3
0.5
0.6
6:00 8:00 10 :00 12 :00 14 :00 16 :00 18 :00
gs
(mo
l m
-2s-1
)
Time (hours)
C
0
1
2
3
4
5
6
6:00 8:00 10 :00 12 :00 14 :00 16 :00 18 :00
VP
D (
leaf-
to-a
ir)
(KP
a)
Time (hours)
Semi -humid region
10/13 /200 6 12 /11 /200 6
10/03 /200 7 12 /18 /200 7
A
0
1
2
3
4
5
6
6:00 8:00 10 :00 12 :00 14 :00 16 :00 18 :00
VP
D (
leaf-
to-a
ir)
(KP
a)
Time (hours)
11/02 /200 6 12 /15 /200 6
09/25 /200 7 12 /19 /200 7
BSemi -arid region
0
2
4
6
6:00 8:00 10 :00 12 :00 14 :00 16 :00 18 :00
E (
mm
ol
m-2
s-1)
Time (hours)
F
0
5
10
15
20
6:00 8:00 10 :00 12 :00 14 :00 16 :00 18 :00
A (
µm
ol
m-2
s-1)
Time (hours)
H
0
2
4
6
6:00 8:00 10 :00 12 :00 14 :00 16 :00 18 :00
E (
mm
ol
m-2
s-1)
Time (hours)
E
0
5
10
15
20
6:00 8:00 10 :00 12 :00 14 :00 16 :00 18 :00
A (
µm
ol
m-2
s-1)
Time (hours)
G
F ), trao t Zonb
(s(t
h
ig. 2. Diurnal variations of vapor pressure deficit (A, B), stomatal conductance (C, Df the year cultivated in field conditions in Agreste (semi-arid climate) and in Foresars indicate standard error.
Table 1), whereas the intrinsic water use efficiency (A/gs) pre-ented a negative correlation with internal CO2 concentration (Ci)
−0.88**, Table 1). We also found a negative correlation betweenhe Ci and photosynthesis (R = −0.34**, Table 1).
The mean leaf SPAD index showed values of 39.9 for the semi-umid region and 42.7 for the semi-arid region, with a statistical
nspiration (E, F) and photosynthesis rate (G, H) of Jatropha curcas at different timese (semi-humid climate) of Brazil. Each point represents the mean of 10 plants, and
difference among the sites (Table 2). The highest SPAD values wereobserved in the month of December 2006 for the semi-humid
region and the month of June 2007 in semi-arid region, and thelowest SPAD values were in December 2007 in both regions withleaf SPAD index of 36.6 and 38.8 in the semi-humid and semi-aridregion, respectively (Table 2).
208 C.M. dos Santos et al. / Industrial Crops and Products 41 (2013) 203– 213
Table 1Pearson’s correlation coefficients between leaf-to-air vapor pressure deficit (VPDleaf-to-air), stomatal conductance (gs), transpiration rate (E) photosynthetic rate (A), internalconcentration of CO2 (Ci), instantaneous water use efficiency (A/E), intrinsic water use efficiency (A/gs), maximum quantum yield of photosystem II (Fv/Fm), leaf tempera-ture − camera temperature (Tl − Tc) in Physic nut (Jatropha curcas L.) cultivated in field conditions in Agreste (semi-arid climate) and in Forest Zone (semi-humid climate) ofBrazil.
Variable E gs A Ci A/E A/gs Tl − Tc
Correlation based on all data (n = 410)VPD(leaf-to-air) 0.02 −0.32** −0.45** −0.01 −0.55** 0.03 −0.18*
E 0.35** 0.55** −0.06 0.01 −0.60 −0.49**
gs 0.32** 0.19* 0.10 −0.14* −0.19**
A −0.34** 0.70** 0.22** −0.25**
Ci −0.55** −0.88** −0.03A/E 0.56** 0.02A/gs 0.08
Correlations based on data with gs ≥0.1 mol m−2 s−1 (n = 312)VPD(leaf-to-air) 0.67** −0.24** −0.22* −0.28** −0.56** 0.24* −0.56**
* Significant difference P < 0.05.** Significant difference P < 0.01.
Table 2Relative chlorophyll content (SPAD values) and effective quantum yield of the photosystem II (˚PSII) of Jatropha curcas L. leaves cultivated in field conditions in Agreste(semi-arid climate) and in Forest Zone (semi-humid climate) of Brazil.
Months SPAD values ˚PSII (12 h)
Semi-humid region Semi-arid region Mean Semi-humid region Semi-arid region Mean
(R = −0.57**), A/gs (R = −0.68**) and a positive correlation withVPD(leaf-air) (R = 0.56**) and SPAD (R = 0.52**) (Table 4).
Table 3Pearson’s correlation coefficients between Photosynthetic rate (A), maximum quan-tum yield of photosystem II (Fv/Fm) and effective quantum yield of the photosystemII (˚PSII) in Jatropha curcas L. leaves cultivated in Agreste (semi-arid climate) and inForest Zone (semi-humid climate) of Brazil (n = 410).
CV % 5.82
eans within the same row followed by the same uppercase letters and means wrobability by Tukey test.
.3. Chlorophyll a fluorescence
In semi-humid region, the maximum quantum efficiency of PSIIFv/Fm) varied little throughout the day, especially in July but, in therier months, there was a small reduction of the Fv/Fm during theay, which occasionally showed a recovery in the afternoon (Fig. 3).nly in the semi-arid region and on the driest month (December006) was observed a Fv/Fm lower than 0.60 throughout the day,ith minimum values of 0.51 near mid-day.
The effective quantum efficiency (˚PSII) differed significantlyetween the semi-humid and semi-arid regions, with averages of.28 and 0.25, respectively (Table 2). In the drier months, par-icularly in December 2006, there was a significant reduction ofhe ˚PSII. The highest value of ˚PSII was found in the semi-humidegion in December 2007. During this period, despite the low soilumidity, the VPD(leaf-air) below 2.0 kPa may have favored the gasxchange, with higher photosynthetic rates throughout the dayFig. 1A and G). There was also a strong positive correction betweenhotosynthesis and ˚PSII (R = 0.60**), and photosynthesis and Fv/Fm
R = 0.55**, Table 3).
.4. Effect of abiotic stress on the organic solutes and antioxidantnzyme
Soluble amino acids (AAs), soluble sugars (TSS) and proline (Pro)
n the leaves of the J. curcas plants did not change among the daysecember 2006, July 2007 and October 2007 (Fig. 4A–C). Yet inecember 2007, there was a significant increase in the content of
oluble amino acids and proline and a reduction of the free sugars
29.36
the same column followed by lower case do not differ among themselves at 5%
compared to the other month. There was also a negative correla-tion between total soluble sugars and amino acids (R = −0.56**), andtotal soluble sugars and proline (R = −0.65**, Table 4).
The AAs had a positive correlation with the physiological param-eters related to photosynthetic efficiency, such as A (R = 0.59**),A/Ci (R = 0.76**), Fv/Fm (R = 0.38*) and ˚PSII (R = 0.58**) (Table 4).Similarly, proline had a positive correlation with photosynthe-sis (A) (R = 0.50**), instantaneous carboxilation efficiency (A/Ci)(R = 0.63**), Fv/Fm (R = 0.42*) and ˚PSII (R = 0.59**), whereas TSS hada negative correlation with A (R = −0.37*), A/Ci (R = −0.51), Fv/Fm
(R = −0.31) and ˚PSII (R = −0.38**).Catalase (CAT) had an increase in activity during the driest
months and in December 2006 and December 2007 (Fig. 4D).The CAT presented a negative correlation with photosynthe-sis (R = −0.37*), stomatal conductance (R = −0.52**), transpiration
Variable Fv/Fm ˚PSII
A 0.55** 0.60**
Fv/Fm 0.54**
** Significant difference P < 0.01.
C.M. dos Santos et al. / Industrial Crops and Products 41 (2013) 203– 213 209
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A
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Fig. 3. Diurnal variations of maximum quantum efficiency of PSII (Fv/Fm) of Jatropha curcas cultivated in field conditions in Agreste (semi-arid climate) and in Forest Zone(semi-humid climate) of Brazil. Each point represents the mean of 10 plants, and bars indicate standard error.
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ig. 4. Free amino acid contents (A), total soluble sugars (B), proline (C), catalase alimate) and in Forest Zone (semi-humid climate) of Brazil. Bars followed by the sa
. Discussion
Northeastern Brazil shows very different characteristics fromhe other regions of the country. The region chosen for study in theorest Zone was in the Coastal Tableland ecosystem region with a
able 4earson’s correlation coefficients between free amino acid (FAA), total soluble sugars (TSSE), stomatal conductance (gs), photosynthetic rate (A), instantaneous carboxilation efficiA/gs), maximum quantum yield of photosystem II (Fv/Fm), effective quantum yield of the phontent (SPAD values) of Jatropha curcas L. leaves cultivated in Agreste (semi-arid climate
* Significant difference P < 0.05.** Significant difference P < 0.01.
(D) in Jatropha curcas L. leaves cultivated in field conditions in Agreste (semi-aridter are not significantly different at 0.05 probability level by Tukey test.
semi-humid tropical climate; the rainfall in this region is influencedby the maritime tropical air mass and cold polar air penetration,
presenting high rainfall values, with an annual mean between 1500and 2000 mm (Souza et al., 2003). While, the semi-arid tropics arelocated in areas that are remote from the ocean and isolated by
), proline (Pro), catalase (CAT), internal concentration of CO2 (Ci), transpiration rateency (A/Ci), instantaneous water use efficiency (A/E), intrinsic water use efficiencyotosystem II (˚PSII), leaf-to-air vapor pressure deficit (VPD) and relative chlorophyll) and in Forest Zone (semi-humid climate) of Brazil (n = 40).
ountainous regions and experience a continuous water shortagehich results from the irregular timeline distribution of rainfall,ith an annual mean of 431.8 mm, from the high evaporation and
ow moisture retention capacity of the vast majority of the soilsn the region (Nascimento et al., 2003). Therefore, these different
eather conditions can interfere with the phenological behaviorSantos et al., 2010) and physiology of J. curcas, thus, altering theultivation strategies, management and crop yield between theseocations.
Under a low VPD(leaf-air), J. curcas maintained a high capacity forhotosynthesis and stomatal conductance in the morning (Fig. 2);ith the increase of the VPD(leaf-air) at mid-day, the plants showed
decrease in both photosynthesis and stomatal conductance. Simi-ar behavior has been found in sugar-apple trees (Annona squamosa.) (Endres, 2007), Eucalyptus clones (Tatagiba et al., 2007) andrapevine (Vitis vinifera) (Yu et al., 2009).
In the present investigation, we observed that J. curcas showed photosynthetic depression at noon. According to Yu et al. (2009),he depression of photosynthesis at mid-day probably results fromoo much light during this period, which can inhibit photosynthesis.ther possible causes of depression at mid-day include an increase
n the vapor pressure deficit of the leaf-air VPDleaf-air (Shirke andathre, 2004; Ohsumi et al., 2008; Oliver et al., 2009) and high tem-erature (Day, 2000; Eamus et al., 2008). The closure of stomatauring mid-day may also decrease the intercellular CO2 concen-ration, inhibiting photosynthesis (Saibo et al., 2009). Thus, theffective regulation of stomatal aperture is critical for the optimalevelopment of plants.
In studies conducted in the field during the dry season with cof-ee plants, Coffea arabica L. (Oliveira et al., 2006) and papaya trees,arica papaya L. (Reis and Campostrini, 2008), the authors have alsoound a reduction in photosynthesis under conditions of a high VPD,robably due to the effect of the VPD on stomatal closure, which
eads to the reduction of internal carbon. Our study also showed negative correlation between VPD and Ci (R = −0.28**) when thetomatal conductance was higher than 0.1 mol m−2 s−1. The reduc-ion of photosynthesis in response to stomatal closure has also beeneported previously in J. curcas under water deficit conditions (Yint al., 2010; Pompelli et al., 2010).
The Tf − Tc showed an inverse correlation with VPDleaf-air, sug-esting that the increase in VPDleaf-air led to an increase inranspiration, causing the cooling of the leaf due to the high latenteat of water vaporization, as has been suggested by Day (2000)een used for the continuous monitoring of the water status in the
eaf and as an indicator of stress.The stomatal conductance of the J. curcas plants showed val-
es close to zero during the driest months (December 2006), whenhere was low soil water availability in both regions, indicating thathe plants were under moderate to severe water deficit during thiseriod. In a situation of moderate water stress, plants keep theirtomata closed to maintain a certain turgor pressure, which is anmportant trait of drought tolerance (Lawlor and Tezara, 2009).
The positive correlation between stomatal conductance andranspiration was observed in this study in response to low soilater availability, showing that transpiration was greatly influ-
nced by stomatal regulation in J. curcas. Similar effects have beenetected in J. curcas seedlings grown in a greenhouse under a watereficit (Dou et al., 2008; Maes et al., 2009a; Pompelli et al., 2010)nd salinity stress (Silva et al., 2010c). This suggests that the partiallosure of stomata is a strong mechanism of J. curcas in response torought tolerance.
Transpiration was negatively correlated with the instanta-
eous water-use efficiency in J. curcas, and similar behavior haslso been observed in other crops (Tatagiba et al., 2007; Endres,007). According to Chaves and Oliveira (2004), in the beginningf water deficit, stomatal conductance decreases faster than the
and Products 41 (2013) 203– 213
photosynthetic rate decrease, which causes an increase in theinstantaneous water use efficiency, whereas the intrinsic wateruse efficiency (A/gs) is negatively correlated with stomatal con-ductance. Several authors have found (in different species) that thereduction in the stomatal conductance has caused an increase in theintrinsic water-use efficiency (Passos et al., 2005; Aasamaa et al.,2010).
The negative correlation between the internal CO2 concentra-tion and photosynthesis suggests that the internal concentrationof CO2 was not limiting for photosynthesis in J. curcas. Accord-ing to Lawlor and Tezara (2009), when combined with a lowwater potential, the increase in the internal concentration of car-bon dioxide causes a progressive reduction in the photosyntheticrate until it becomes zero. Among the causes for this effect, thedecrease in carboxylation efficiency caused by the direct inhibitionof the photosynthetic apparatus has been cited (Shirke and Pathre,2004; Oliver et al., 2009). However, studies have reported that, atnoon, the carboxylation efficiency has decreased, whereas the Ciremained constant (Yu et al., 2009).
An increase in internal CO2 concentration due to stress couldbe happed in response to stomatal closing, but it is not a universalresponse, as there have been reports of both increases (Yin et al.,2010) and decreases (Dou et al., 2008) of the internal CO2 concen-tration in J. curcas when the plants have been subjected to waterdeficit. According to Oliver et al. (2009), the response of the stom-ata to changes in the internal CO2 concentration are also stronglydependent on other variables, such as light intensity, plant waterstatus, temperature and vapor pressure deficit. These abiotic factorsprobably do alter the internal CO2 concentrations.
The highest SPAD values of J. curcas leaves were found duringthe months of December 2006 (dry season) for the semi-humidregion and June 2007 for the semi-arid region (the rainy season),a period during which the highest numbers of young leaves werefound (data not shown). According to Ruiz-Espinoza et al. (2010)the SPAD index depends on several factors, including the speciesstudied, the diversity of the leaves, the thickness of the leaves andthe leaf age. A study conducted in J. curcas under field conditionshas found that plants with increased production of new leaves had ahigher SPAD index, with values over 40, and higher photosyntheticrates (Yong et al., 2010).
A consequence of the reduction of photosynthesis caused byenvironmental stresses, such as high radiation and water deficit,is the exposure of the plant to excess energy, which, if not safelydissipated, may cause changes in the functional state of the thy-lakoid membranes of the chloroplasts. This can cause changes inthe characteristics of fluorescence signals, which can be quan-tified in the leaves, providing data to estimate the inhibitionor damage in the process of electron transfer in photosystem II(Baker and Rosenqvist, 2004; Pospisil, 2009). Studies conductedon J. curcas have shown the use of the fluorescence emissionkinetic parameters in order to detect damage caused by differenttypes of stress (Liang et al., 2007; Zheng et al., 2009; Silva et al.,2010c).
In J. curcas, the variation of the Fv/Fm throughout the day variedless in the semi-humid region in relation to the semi-arid region.In the semi-humid region, the Fv/Fm ranged from 0.81 to 0.76,and in the semi-arid region, the Fv/Fm was between 0.79 and 0.50.According to Bolhàr-Nordenkampf et al. (1989), when a plant’s pho-tosynthetic apparatus is intact, its values of Fv/Fm vary between0.75 and 0.85, and values below 0.75 indicate a stressful situationand, thus, a reduction of the photosynthetic potential of the plant(Maxwell and Johnson, 2000). Studying the effect of photochemical
activity of J. curcas under saline and water stress, Silva et al. (2010c)has observed that the Fv/Fm activity was not changed and main-tained average values of 0.85 during stress. In contrast, anotherstudy (Dou et al., 2008) has found a decrease in the Fv/Fm ratio
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nder a water deficit. Studying J. curcas seedlings subjected to coldtress, Liang et al. (2007) has observed Fv/Fm values of 0.95 underormal conditions and values below 0.7 during stress. In addition,heng et al. (2009) has found average Fv/Fm values of 0.85 in J. curcasn the absence of stress.
During the driest period, December 2006, the leaves of the J.urcas plants showed a marked decrease in the Fv/Fm ratio at mid-ay. In the semi-humid region, the minimum value observed was.73, and it recovered by the end of the day, indicating that thereas a process of dynamic photoinhibition. Such dynamic pho-
oinhibition usually occurs during the hottest hours of the day,nder conditions of a moderate excess of light, which involves
transformation of light energy into heat, causing a decrease inhe quantum efficiency (Long et al., 1994; Casaroli et al., 2007).n this way, a reversible portion of photosynthesis related tohotoinhibition should not be seen as damage but as a protec-ive mechanism that allows dissipating excess thermal energyCampostrini, 1997). In the semi-arid region, the minimum val-es of the Fv/Fm were close to 0.5 around noon and not recoveredvernight, indicating that there was a chronic photoinhibition inhese plants. This chronic photoinhibition involves prolonged peri-ds of leaf exposure to high levels of solar radiation, thus reducinghe quantum efficiency and the maximum photosynthetic rateCasaroli et al., 2007). Under severe water stress, plants oftenhow a marked photoinhibitory effect, characterized by a signif-cant decrease in the quantum yield potential. In this case, the
ater deficit, in combination with high irradiance levels, can cause significant reduction in the efficiency of photosynthesis (Longt al., 1994; Lemos-Filho, 2000).
In the present study, a reduction in the effective quantum effi-iency (˚PSII) was found during the driest months, demonstrating
lower utilization of the light energy. According to Maxwell andohnson (2000), a reduction in the ˚PSII is a physiological indexuitable for the evaluation of photosystem II efficiency in vivo, inhis case, indicating a low proportion of energy absorbed. In young. curcas plants under water and saline stress (Silva et al., 2010c)nd cold stress (Zheng et al., 2009) was found reductions in effec-ive quantum efficiency (˚PSII). The authors have suggested thathis decrease in the intensity of photosynthesis is one mechanismf photoprotection used by the plant to preserve the photochemicalpparatus during stress.
The accumulation of organic solutes, such as soluble aminocids and proline, in J. curcas was higher during the dry season, inecember of 2007. This may be part of a mechanism that prevents
oss of water in the plant through osmotic adjustment. The increasef these solutes coincided with low rates of the plant photosynthe-is in response to abiotic stress in each region. These responsesorroborate the studies conducted on J. curcas grown under watereficit conditions in a greenhouse (Silva et al., 2010b) which hasound increased synthesis of soluble sugars and amino acids withhe increase of a water deficit which was associated with low sto-
atal conductance and transpiration. On the other hand, Pompellit al. (2010), in a fast dehydration experiment, has found lower con-entrations of soluble amino acids during water stress which wasssociated with a decrease of stomatal conductance. According tohou and Yu (2010), the accumulation of metabolites in the plantnder water stress can act as compatible osmolytes, maintainingell turgor at lower water potentials.
In a study conducted in castor bean (a species of the same fam-ly of J. curcas) under water stress, the accumulation of proline,oluble sugars and soluble amino acids has been observed in aenotype considered to be tolerant to drought (Babita et al., 2010).
urthermore, a positive correlation between proline accumulationnd tolerance to water stress has been shown in different speciesClaussen, 2005; Valliyodan and Nguyen, 2006; Guo et al., 2010),ulfilling a key role in osmotic balance as well as an antioxidant
and Products 41 (2013) 203– 213 211
agent in plants that are tolerant to drought (Cordeiro et al., 2009;Valliyodan and Nguyen, 2006).
A limitation of photosynthesis may occur in the plant underdrought conditions, inducing oxidative stress due to excess energy,which, if not safely dissipated, may cause an over-excitation ofthe reaction centers of photosystem II, increasing the productionof ROS in the chloroplasts (Carvalho, 2008). In order to mitigatethe oxidative damage caused by ROS, plants possess a complexantioxidant defense system involving non-enzymatic and enzy-matic antioxidants (Scandalios, 2005). Catalase plays a crucial rolein the antioxidant system, as it operates in the dismutation ofhydrogen peroxide (H2O2) into oxygen and water (Asada, 2006).This enzyme is considered very sensitive to conditions of abioticstress and serves as a marker of stress (Gao et al., 2008). Severalauthors have shown that CAT activity increases in J. curcas sub-jected to water deficit (Silva et al., 2010a; Pompelli et al., 2010) andthese effects have also been observed for salinity stress (Gao et al.,2008). In our study was observed an increase in CAT activity duringthe dry season, a period with low soil water availability, high VPDand light. Therefore, this enzyme can be considered very sensitivemarker to abiotic stress conditions and has proven to be an effectiveindicator of physiological stress in J. curcas.
5. Conclusions
The higher vapor pressure deficit of air (dry season) may beinvolved in stomatal closure in J. curcas, which contributed to areduction in the values of the net photosynthetic rate.
J. curcas showed a dynamic photoinhibition for most of the daysobserved, even in dry season; the plant only showed chronic pho-toinhibition under a severe water deficit.
The accumulation of organic solutes, such as proline and solubleamino acids and, especially, the increase in CAT activity, duringthe period of water deficit revealed an important mechanism ofadaptation of J. curcas in response to drought.
Acknowledgments
We acknowledge the Research Foundation for the State ofAlagoas (FAPEAL) and the National Council for Scientific and Tech-nological Development (CNPq) for financial support.
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