Jatropha curcas L. (Euphorbiaceae) modulates stomatal traits in response to leaf-to-air vapor pressure deficit

Biomass and Bioenergy 81 (2015) 273e281

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Research paper

Jatropha curcas L. (Euphorbiaceae) modulates stomatal traits in response to leaf-to-air vapor pressure deficit Bety S. Hsie a, Keila R. Mendes a, Werner C. Antunes b, Laurício Endres c, Mariana L.O. Campos a, Felipe C. Souza c, Nivea D. Santos a, Bajrang Singh d, Emília C.P. Arruda e, Marcelo F. Pompelli a, * a

Plant Ecophysiology Laboratory, Federal University of Pernambuco, Department of Botany, CCB, Recife, PE, 50670901, Brazil
, Maringa
, PR, Brazil Department of Biology, University of Maringa
, AL, Brazil Plant Physiology Laboratory, Federal University of Alagoas, Center of Agronomy, Maceio d National Botanical Research Institute, Rana Pratag Marg, Lucknow, India e Plant Anatomy Laboratory, Federal University of Pernambuco, Department of Botany, Recife, PE, Brazil b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 November 2014 Received in revised form 3 July 2015 Accepted 12 July 2015 Available online xxx

Cultivation of Jatropha curcas in arid and semiarid non-cultivated areas could be a sustainable strategy for stimulating biofuel production without competing with food crops for land and water resources. J. curcas is considered a drought-tolerant species; however, the mechanisms that provide tolerance are unknown. Few efforts have been made to understand the connections between stomatal development and environment conditions. Here, we compared changes in stomatal density (SD) and stomatal index (SI) and their influence on gas exchange in J. curcas. Plants were cultivated in both rainy and dry regions. We describe a distinctive distribution of stomata under the adaxial and abaxial leaf epidermises, where higher SD may have caused the increase in stomatal conductance (gs) with positive effects on net photosynthetic rate (PN). However, when rain was excluded, the variation in gs was strongly related to vapour pressures deficit (VPD), and VPD was strongly related to the PN. Thus, our results suggest that J. curcas may also contribute mitigating the effect of CO2 deposition in the atmosphere, given that a remarkable change in SD and other leaf traits was observed in response to seasonal variations. Moreover, multivariate analysis highlights the high sensitivity of J. curcas plants to VPD which in turn induces rapid stomatal closure and consequent reduction of PN for long periods of time which reflect into a change in the pattern of development resulting in higher SI. These results can help us to understand the relationship between stomatal features and gas exchange in response to environment changes. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Canonic correlation Gas exchange Purging nut Stomata density Stomata index

1. Introduction Jatropha curcas L., purging nut or physic nut, is a large perennial plant in the Euphorbiaceae family. The species is a large deciduous shrub or small tree, with a life expectancy of up to 50 years [1]. In recent decades, J. curcas has become popular because of its properties and uses. J. curcas seeds contain approximately 25e58% oil

Abbreviations: CCA, canonical correlation analysis; Ci:Ca, ratio of the internal-toambient CO2 concentration; gs, stomatal conductance; LA, leaf area; OD, ordinary cell density; PN, net photosynthetic; RH, relative humidity rate; SD, stomatal density; SI, stomatal index; VPD, leaf-to-air vapor pressure deficit; WUE, water use efficiency; WUEi, intrinsic water-use efficiency. * Corresponding author. E-mail address: [email protected] (M.F. Pompelli). http://dx.doi.org/10.1016/j.biombioe.2015.07.014 0961-9534/© 2015 Elsevier Ltd. All rights reserved.

[2,3], that can be extracted and converted to biodiesel. The oil can also be used as cooking or lighting fuel, medicine, a biopesticide, and for soap making. Oil extraction by-products can be used as organic fertilizer, combustible fuel, or for biogas production [4]. Production of J. curcas as an energy crop has been proposed as a sustainable use of non-cultivated and marginal areas in arid and semiarid regions [5,6] characterized by high evaporative demand and low water availability [7]. Although all recent studies confirm the high drought resistance of J. curcas [8e10], there is a lack of information on leaf traits particularly on the effect of seasonal changes in leaf water potential on stomatal functioning and photosynthesis characteristics. The ability of species to respond to a new environments is critical for growth and survivals [11]. Leaf structures reflect the effects of water deficit more clearly than stems or roots [12].

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2. Material and methods 2.1. Study site and plant material The climate of the state of Alagoas is influenced by weather systems of the intertropical convergence zone and the easterly waves [28], causing highly variable rainfall across the state. The Caatinga ecosystems covers 52% of Alagoas; and is an arid-tosemiarid ecosystem that occupies the region between the Amazon forest and the Atlantic rainforest, covering 735,000 km2 of northeast Brazil where lives around 28 millions of people [29,30]. This ecosystem includes rainy climates near the Atlantic rainforest

and desert-like arid climates in the Caatinga [31]. Regions near the Atlantic rainforest receive more than 4000 mm year
1. In contrast, the Caatinga has high rates of potential evapotranspiration (1500e2000 mm annually) and low rainfall (300e1000 mm annually) that is usually concentrated over 3e5 months [32]. This study was conducted on 5-year old J. curcas plants, grown in experimental fields in a semi-humid region near the city of Rio Largo (09º280 4200 S, 35º5102100 W, 39 m a.s.l.) and in a semiarid region near the city of Igaci (09º3201300 S, 36º380 0100 W, 240 m a.s.l.) (see Ref. [2] for detailed sites description). Compared with Igaci, Rio Largo receives more precipitation annually, has a shorter, less severe dry season and has higher monthly precipitation in both seasons (Fig. 1A). Annual precipitation during the study year (2007) was within the range of historical means of 1700e1900 mm year
1 for Rio Largo and 800e1000 mm year
1 for Igaci [33]. In both areas, the rains were concentrated mainly in the autumn and winter months from March to August. In both ecosystems, water deficit is caused by soil water deficits and high atmospheric VPD [2,34]. 2.2. Stomatal characteristics and leaf area To ensure that the leaves used in this study had grown during this experiment, all leaves used for morphological evaluations were marked from the primordial stage to full expansion (when sampled). Within each population described above, a fourth

350

30

Rainfall (mm)

300

20

200

15

150

10

100 50

5

0

0

B

4 AET (mm)

25

A

250

*

*

Temperature (ºC)

Stomatal responses are essential for the acclimation of plants to new environmental conditions and are thus important determinants of plant survival [13,14]. Generally, adaptation of plants to drought requires transitory and long-term mechanisms for maintaining high water potential by increasing water absorption and reducing transpiration [15]. Excessive water loss by transpiration as a result of a high VPD may induce stomatal closure. However, gas exchange in higher plants is mainly determined by both stomatal opening (pore aperture) and stomatal anatomical features (size and density) [16]. Consequently, the effect of environmental features on leaf conductance involves both long-term processes and short-term dynamics. Short-term events are mediated by adjustments in pore aperture and are reversible, while long-term processes refer to leaf expansion and involve stomatal size and ~ ber [19] compared the effects of density [17,18]. Aasamaa and So different environmental conditions and found that the stomata of six temperate deciduous tree species grown in a greenhouse responded more strongly to changes in hydraulic environmental factors, e.g., air humidity and water supply than to changes in photosynthetic environmental factors, e.g., light intensity and air CO2 concentration. Moreover, in field conditions, several environmental factors converge to change stomatal morphology and physiology [13]. Stomatal diffusion resistance, and hence conductance, is somehow directly related to the size and spacing of stomata on the leaf surface, i.e., a trade-off exists between the size and number of stomata [20]. Most studies of stomatal morphology have focused on the closure mechanism because it is involved in water loss, particularly when plants are grown in high relative humidity (RH) [21]. Other anatomical features involved in water loss have received meager attention. For instance, a negative correlation between stomatal size and stomatal functioning has been described in some species [14,22e24]. However, at the leaf level, gs is not solely determined by stomatal pore aperture but also depends on stomatal density (SD), pore length and pore depth [21]. Variation in stomatal size will therefore largely determine optimization of water flux under water-limiting conditions [17]. Photosynthesis is particularly sensitive to water deficits, because stomatal closure restricts CO2 influx and reduces carbon gain as soil water status decreases [8,25]. Understanding how crops in arid and semiarid regions respond to changes in leaf-to-air VPD is important because some climate models predict an increase in temperature and a decline in rainfall in coming decades [26,27], which may affect plant functioning. The objectives of this study were therefore to determine the response patterns of SD and stomatal index (SI) to different degrees of water restriction and to determine the relationship with gas exchange of J. curcas grown in semiarid and semi-humid ecosystems. Thus, we aimed to clarify two questions: (i) Does variation in seasonality affect leaf and stomatal traits and carbon gain in saplings of J. curcas in semiarid and semi-humid ecosystems? (ii) Does the increase in the number of stomata per unit area (i.e., SD) affect gas exchange of this species?

* *

3 2

* *

*

1 0 Water soil availability (mm)

274

200

*

C

*

* *

150

*

* * *

100 50

*

* 0

J

F M A M J

*

J A S 0 N D

Fig. 1. Climatic characteristics: (A) total monthly rainfall (bars) and mean temperatures (lines); (B) actual evapotranspiration rate (AET) and (C) soil water balance (store) from January to December 2007 in the tropical dry (black symbols) and tropical semihumid (white symbols) ecosystems. Means followed by asterisks represent statistically significant differences within each month. The values represent the media (±SE), n ¼ 30. Source: Agritempo [79].

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expanded leaf from the apex of five plants were collected in the rainy season (July 2007) or in the dry season (December 2007) for stomatal counts. Small pieces of each leaf sample (c. 6 mm square) were collected from a central position and were fixed [35]. The selected leaves were also used for gas exchange measurements (see below). All leaf samples were then treated at 60  C for 2 h with a Franklin solution [36] composed of 1:1 volume of glacial acetic acid and hydrogen peroxide. The tissues were then rinsed three times with distilled water, stained with 10 g L
1 safranin in 1:1 volume mixture of ethanol and water and mounted on glass slides with a 1:1 glycerin:water mixture [37]. The stomata were counted under a microscope at 10  magnification using twenty different fields of view for five different leaves per locale (i.e., 100 different fields of view). Each field of view consisted of 0.6 mm squares registered in a Leica DM IRB microscope fitted with a Leica DC 300F camera (Leica Microsystems, Wetzlar, Germany). The number of stomata per visual field of view was converted to the number of stomata per square mm, hereafter referred to as stomatal density. The stomatal index was estimated as described earlier Pompelli, Martins [38]. For the leaf area (LA) measurements, 100 fully expanded leaves were harvested in the rainy and dry seasons at the two sites described above. Leaves were kept in airtight bags and sent to the laboratory, where were measured leaf area as described previously for J. curcas [39]. 2.3. Gas exchange measurements Gas exchange was measured on one mid-canopy, healthy, fully sun-exposed leaf per tree from 10 trees per site. Net photosynthesis (PN) and stomatal conductance (gs) were measured with an openpath system, using a portable infrared gas analyzer with a 6 cm2 leaf cuvette (IRGA; LCProþ, ADC Ltd. Bioscientific, Hoddesdon, UK) under ambient temperature, light and CO2 concentration (380 mmol mol
1). The intrinsic water-use efficiency (WUEi) was calculated as the ratio between PN and gs. Data were collected between 08:00 and 09:00 h in the rainy season (July 2007) or in the dry season (December 2007). 2.4. Statistical analysis Five replicates were used to determine morphological characteristics. For all other analyses, ten replicates were used, with each replicate consisting of one J. curcas tree. Normality was tested with a ShapiroeWilk test [40] and homogeneity of variance was tested with a BrowneForsyth test [41]. All analyses were conducted with Statistica version 7.0 (StatSoft, Tulsa, OK, USA). All data are presented as means plus standard errors (SE). All results were analyzed with a mixed-model ANOVA, and means were compared using an SNK test with the statistical software package SigmaPlot version 11.0 (Systat Software Inc., Chicago, USA). The parametric correlations were analyzed with the statistical software Statistica version 7.0 (StatSoft, Tulsa, OK, USA). The results were considered significant at p  0.05. To better understand the influence of leaf morphology on the physiological traits, a canonical correlation analysis (CCA) was conducted [42]. All correlations were analyzed with the statistical software package Statistica version 7.0 (StatSoft, Tulsa, OK, USA). The results were considered to be significant at p  0.05. 3. Results 3.1. Stomatal characteristics and leaf area Irrespective of region, the dry season prompted a decrease in the individual LA, relative to the rainy season (Table 1). Therefore,

275

Table 1 Leaf area (cm2) of the Jatropha curcas L. plants grown in tropical dry and tropical semi-humid ecosystems in two different seasons. Significant seasonal differences (column) among the means are represented by different capital letters, and significant differences between populations within the same line are represent by lower case letters (p  0.01, NewmaneKeuls test). The values represent the media (±SD). n ¼ 100. Water regimes

Tropical dry

Tropical semi-humid

Rainy season Dry season

73.66 ± 5.14 Aa 42.33 ± 1.86 Ba

63.97 ± 3.63 Aa 45.29 ± 1.78 Ba

epidermal cells from the leaves collected in the dry season were smaller than those from leaves collected in the rainy season, as indicated by the increase in density of ordinary cells (Fig. 2A, D). This pattern lead to a negative relationship between SD and LA (r ¼
0.671; p  0.001). Nevertheless, there were reductions of SD with increases in rainfall (Figs. 1 and 2). Thus, the results address the effects of ecological parameters such as precipitation and VPD on the micromorphological features of leaf stomata. Abaxial and adaxial epidermal cells of J. curcas were typically polygonal with straight or curved anticlinal walls (Figs. 3 and 4). The leaves were amphistomatic, with paracytic-type stomata deeply inset under circular rims formed by 5e6 epidermal cells (Figs. 3 and 4). The leaves of J. curcas possess stomata with distinctive arrangements on the adaxial (Fig. 3) and abaxial (Fig. 4) epidermal surfaces. Twelve to fifteen times more stomata (i.e., larger SD) exist on the abaxial epidermis than on the adaxial in the rainy season; while only three to five times more stomata were verified in the dry season (Fig. 2). On the adaxial surface, the SD of the tropical dry leaves ranged from 11 to 52 stomata per square mm in the rainy to dry seasons, respectively. On the abaxial surface, the SD ranged from 135 to 258 stomata per square mm in the rainy to dry seasons respectively. For the semi-humid leaves, the SD seemed to have the same pattern as in the dry tropical climate leaves, although with less variation between seasons (Fig. 2). Therefore, SD and SI seemed to be affected by cell expansion, as shown by the positive correlation between OD and SD (r ¼ 0.805; p  0.0001) or SI (r ¼ 0.488; p  0.0001). However, the p-values were higher when the abaxial surface data were analyzed without the adaxial data. Similarly, the difference was more pronounced when dry season values were analyzed without the rainy season data. Additionally, the occurrence of abnormal stomata in the dry season was higher (28.5 ± 2.7%) on the adaxial surface of leaves collected in semiarid regions than on leaves from the other treatments, which did not differ from one another (Fig. 5). 3.2. Gas exchange measurements PN and gs were higher in the wet season than in the dry season in plants grown at the dry tropical site. However, the opposite was observed in plants grown in semi-humid conditions (Fig. 6). The gs followed the same trend as PN; however, the gs decreased more than the PN at the dry tropical site (Fig. 6). The differences in PN and gs of J. curcas plants grown in the semi-humid and semiarid regions reflected the varying climatic conditions, with higher soil water availability (Fig. 1C), lower VPD (Fig. 6F) and lower evapotranspiration (Fig. 1B) during the rainy season than during the dry season. For example, in the dry tropical plants, PN and gs decreased 85% and 97%, respectively, when plants were subjected to drought. Under water deficit, PN and gs of the semi-humid tropical plants increased 23.5% and 40.7%, respectively, compared with the rainy season plants. This indicates that the reduction in irradiance because of cloudiness during the rainy season was the main factor limiting carbon uptake. There was a strong relationship between PN and gs

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B.S. Hsie et al. / Biomass and Bioenergy 81 (2015) 273e281

Ordinary cell density (mm )

1800

A

1200

Ab

Aa

Bb

1200

Ba

Ab

600

1800

Aa

D

Bb

Ba

600

0

0

Aa

E

Stomatal density (mm )

B

Ab

250 200

Ba Ba

150 100 50

F

Stomatal index (%)

C

0 Aa

Aa

Ba

Aa

10

Aa Ab

Rainy season

Dry season

15

5

Rainy season

Dry season

0

Fig. 2. Ordinary cell density (A, D), stomatal density (B, E) and stomatal index (C, F) as measured in adaxial (A, B, C) and abaxial (D, E, F) surface of epidermis of leaves of field-grown Jatropha curcas plants. These measurements were made on plants grown in the tropical dry (black symbols) and tropical semi-humid (white symbols) ecosystems from wellexposed leaves in two different seasons. Significant seasonal differences among the means are represented by different capital letters, and significant differences between populations within the same month are represented by lower case letters (p  0.01, NewmaneKeuls test). The values represent the media (±SE). n ¼ 5.

during both the rainy and dry seasons (r ¼ 0.880; p  0.001), confirming that photosynthetic rates (PN) were strongly dependent on gs. The decrease in PN and gs were seemingly caused by the decrease in VPD, an observation confirmed by the correlation between VPD and PN (r ¼
0.477; p ¼ 0.05) and gs (r ¼
0.528; p ¼ 0.001). It is worth noting that the VPD was higher during the dry season, with values up to 4 kPa and a minimum of 2 kPa in both ecosystems (Fig. 6F). An increase in the leaf SD was strongly correlated with a decrease in the leaf area (r ¼
0.671; p ¼ 0.001). Thus, smaller leaves with more stomata must have been one of the causes of the reduction in gas exchange, as evidenced by the negative correlation between SD and PN (r ¼
0.788; p  0.001) and SD and gs (r ¼
0.761; p  0.001). Furthermore, a positive correlation was verified between SD and WUEi (r ¼ 0.790; p  0.001) (Table 2). Taken together, these results indicate that a decrease in the leaf area is associated with higher SD and is closely linked with higher leaf WUEi because gs decreased more than PN (Fig. 6). The CCA analysis corroborates the Pearson (c.a., simple, linear) correlations. Thus, regardless of the ecosystem studied, the morphological variables present distinct correlations only with the first axis of the CCA. SD was negatively correlated and SI was positively correlated with morphological features. For the semihumid tropical regions, the CCA between the morphological and the physiological variables was significant (p value ¼ 0.05) for the canonical pair, with a high correlation coefficient (r ¼ 0.993) for the

first axis (Table 3). These results suggest that SD (cc ¼
1.191) had a positive correspondence with VPD (cc ¼
0.835) and leaf area (cc ¼
0.274). The Ci:Ca ratios had contrasting relationships with SD and SI, while Ci:Ca ratio (cc ¼ 0.375) was negatively associated with SD (cc ¼
1.191), a positive relationship is associated with SI (cc ¼ 0.636). In the dry tropical ecosystem, the physiological variables were largely unrelated to the variation in SD but strongly correlated with SI. Thus the results suggest that SI (cc ¼ 0.497) was positively associated with PN (cc ¼ 1.623), VPD (cc ¼ 0.609), gs (cc ¼ 0.376) and WUEi (cc ¼ 0.362). In these plants, the canonical correlations between the morphological and the physiological variables were also significant (r ¼ 0.997; p  0.01). 4. Discussion The decrease in leaf area observed in this study during the dry season corroborates the findings of Shen, Pinyopusarerk [43], who used dendrogram analysis to reveal high genetic diversity and plasticity in J. curcas. The reduction in leaf area and plant growth allows plants to reduce their transpiration, and increase water use efficiency (WUE). The decrease in SD in the rainy season is closely related to leaf growth; smaller leaves should have higher stomatal densities, mainly due to the packing of the epidermal cells and the reduction in epidermal cell expansion. This is in agreement with Beerling and Chaloner [44], who describe widely spaced stomata and lower SD in Quercus robur leaves that developed during the

B.S. Hsie et al. / Biomass and Bioenergy 81 (2015) 273e281

277

Fig. 3. Drawings of leaf adaxial epidermis surfaces in Jatropha curcas plants grown under tropical semi-humid (A, B) and tropical dry (C, D) ecosystems from well-exposed leaves in the rainy (A, C) or dry (B, D) seasons. All images were processed to a fully expanded, sunlit, upper canopy leaves from plagiotropic branches. Bars 100 mm.

rainy season. The amphistomatic pattern of J. curcas probably confers some adaptive advantage to the plant. According to Fahn and Cutler [45],

amphistomaty is common in woody species because these species typically occur where CO2 may limit photosynthesis (sunny environments). This feature facilitates the diffusion of CO2 and leaf

Fig. 4. Drawings of leaf abaxial epidermis surfaces in Jatropha curcas plants grown under tropical semi-humid (A, B) and tropical dry (C, D) ecosystems from well-exposed leaves in the rainy (A, C) or dry (B, D) seasons. All images were processed to a fully expanded, sunlit, upper canopy leaves from plagiotropic branches. Bars 100 mm. Note the details of defective stomata (B, D; bars 20 mm).

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B.S. Hsie et al. / Biomass and Bioenergy 81 (2015) 273e281

Abnormal stomata (%)

36 Aa

30 24 18 12 6 0

Ab Ba

Aa

Semi-humid

Dry

Fig. 5. Abnormal stomata as computed in dry season leaves of field-grown Jatropha curcas plants. These measurements were made on adaxial (black symbols) and abaxial (white symbols) surfaces of epidermises. The plants grown in the tropical dry and tropical semi-humid ecosystems from well-exposed leaves. Significant populations differences among the means are represented by different capital letters, and significant differences between epidermal surfaces within the same population are represented by lower case letters (p  0.001, NewmaneKeuls test). The values represent the media (±SE). n ¼ 5.

cooling [11,46,47]. The amphistomatic pattern of J. curcas was also reported by Olowokudejo [48]. Interestingly, even with an amphistomatic pattern, the SD of the adaxial and abaxial epidermis differs.

Lower stomatal density on the adaxial surface of the leaf epidermis can be very important to save water, particularly during periods of high water deficit [49,50]. Salisbury [51] states that SD on upper and lower surfaces tends to ‘augment or diminish in a parallel manner’, depending on growth conditions. The data presented in this study do not corroborate this pattern because the adaxial and abaxial stomatal densities responded differently to the dry season. For instance, SD in adaxial surfaces on dry-season leaves were 5e6 times higher than that on rainy-season leaves; on the abaxial surfaces, this ratio was 1.3e2.0 times higher. Stomata of J. curcas are encrypted, sunken and covered. These crypts have a role in facilitating CO2 diffusion from the abaxial to adaxial palisade cells [52], mainly in dry climates where reduced transpiration is an important feature to save water, this provides circumstantial evidence of the xeric conditions experimented by J. curcas leaves [53]. J. curcas has paracytic-type stomata. However, some studies have documented other types of stomata, i.e., anisocytic and anomocytic [54], anomocytic only [55] or paracytic and anisocytic [56]. There is no evident explanation for these differences, which may be due, in part, to the natural variation that is expected in these geographically widespread species. A possible source of error is the different interpretation of stomatal types by different workers. Regardless, paracytic stomata are the most prevalent in J. curcas and are regarded by Raju and Rao [54] as the standard type for the family Euphorbiaceae.

Fig. 6. (A) Net Photosynthesis (PN), (B) stomatal conductance (gs), (C) intrinsic water-use efficiency (WUEi), (D) internal-to-ambient CO2 concentration (Ci/Ca), (E) leaf temperature (Tleaf) and (F) leaf-to-air vapor pressure deficit (VPD) as measured in leaves of field-grown Jatropha curcas plants. These measurements were made on plants grown in the tropical dry (black symbols) and tropical semi-humid (white symbols) ecosystems from well-exposed leaves between 08:00 and 09:00 h in two different seasons. Significant seasonal differences among the means are represented by different capital letters, and significant differences between populations within the same month are represented by lower case letters p  0.01, NewmaneKeuls test). The values represent the media (±SE). n ¼ 10.

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279

Table 2 Pearson's coefficient between morphological and physiological features in Jatropha curcas L. plants grown in tropical dry and tropical semi-humid ecosystems in two different seasons. LA ¼ leaf area (cm2), SD ¼ stomatal density (mm
2), SI ¼ stomatal index (%), OD ¼ ordinary cell density (mm
2), PN ¼ net photosynthesis (mmol CO2 m
2 s
1), gs ¼ stomatal conductance (mmol H2O m
2 s
1), VPD ¼ leaf-to-air vapor pressure deficit (kPa), Ci:Ca ¼ ratio of the internal-to-ambient CO2 concentration, WUEi ¼ intrinsic water-use efficiency (mmol CO2 mol
1 H2O). Values followed by *, **, and ***, represent significance differences at p  0.05, p  0.01, and p  0.001. Means followed by ns do not represent significance differences at p  0.05. Features

LA

SD

SI

OD

PN

gs

VPD

Ci:Ca

WUEi

LA SD SI OD PN gs VPD Ci:Ca WUEi

e
0.671*** 0.252ns
0.414* 0.361ns 0.428*
0.394ns 0.367ns
0.419ns


0.671*** e
0.340ns 0.805***
0.788***
0.761*** 0.828***
0.462* 0.790***

0.252ns
0.340ns e 0.488*** 0.537** 0.514**
0.331ns 0.077ns
0.457*


0.414* 0.805*** 0.488*** e
0822***
0.794*** 0.837***
0.427ns 0.831***

0.361ns
0.788*** 0.537**
0.822*** e 0.880***
0.477* 0.143ns
0.724***

0.428*
0.761*** 0.514**
0.794*** 0.880*** e
0.528*** 0.236ns
0.770***


0.394ns 0.828***
0.331ns 0.837***
0.477*
0.528*** e
0.555** 0.733***

0.367ns
0.462* 0.077ns
0.427ns 0.143ns 0.236ns
0.555** e
0.326ns


0.419ns 0.790***
0.457* 0.831***
0.724***
0.770*** 0.733***
0.326ns e

In this study, we showed that water deficit affected both SD and SI, suggesting that the water deficit alters not only the expansion of stomatal and nonstomatal cells but also stomatal frequency, particularly on the adaxial epidermis (Fig. 2). Royer [57] studied one hundred species and found that 36% showed responses to environmental CO2. The plants responded to the CO2 trend by reducing SD and SI as atmospheric CO2 levels increased over geologic time scales. Furthermore, whether under laboratory conditions or in open-top chambers, SD and SI generally respond inversely to CO2 levels [58,59]. Thus, CO2 appears to inversely affect stomatal formation, although the mechanism may involve CO2 perception, signaling and genetic adaptation that occurs over a longer period of time [57]. We also believe that exposing Jatropha plants to water deficits induces stomatal closing and presumably reduced intercellular CO2 concentrations. This may be followed by low internal CO2 perception, a signal from mature leaves might be transmitted to the leaf primordia [60], and the genes may be modulated to induce stomatal differentiation in young leaves [61]; this could result in a three-to five-fold increase in the SI (Figs. 2C, 3 and 4).

4.1. Gas exchange measurements Among plants grown in the dry tropical region, the low PN in the dry season could reflect either low soil water availability or the high VPD during the dry months. Stomatal response and sensibility to

Table 3 Coefficients of canonical correlations between the morphological features and the physiological features for tropical dry and tropical semi-humid ecosystems. Variables

Morphological features SDabaxial (mm
2) SIabaxial (%) Physiological features Leaf area (cm2) VPD (kPa) gs (mmol H2O m
2 s
1) PN (mmol CO2 m
2 s
1) WUEi (mmol CO2 mol
1 H2O) Ci:Ca Canonical R Significance

Tropical semi-humid

Tropical dry

CC1

CC2

CC1

CC2


1.191 0.636

0.014
1.007


0.527 0.497


2.282
2.289


0.274
0.835
0.181 0.055
0.045 0.375 0.993 0.050


0.467
0.477 0.314 1.071 0.145 0.827 0.445 0.963


0.070 0.609 0.376 1.623 0.362
0.016 0.997 0.013


1.278 5.542 6.346
2.490
1.681 1.187 0.555 0.894

CC1, CC2 coefficients of canonical correlations for the first and second canonical axis, respectively. SDabaxial stomatal density of abaxial surface of epidermis, SI stomatal index of abaxial surface of epidermis, VPD leaf-to-air vapor pressure deficit, PN net CO2 assimilation rate, WUEi intrinsic water use efficiency and Ci:Ca internal-toambient CO2 concentration.

high VPD in different species are well documented in the literature, particularly in semiarid environments [62,63]. A possible explanation for the increase in PN and gs of J. curcas plants grown in the semi-humid region during dry season is that higher irradiance in the dry period outweighed the negative effect of the increase in VPD on stomatal aperture. However, the decline in soil water content during the dry season does not seem to have been large enough to have reduced photosynthesis in plants grown in the dry tropical region. The close correlation between PN and gs (r ¼ 0.880; p  0.001, Table 2) confirms that photosynthetic rates are strongly dependent on gs, as previously reported in J. curcas [7,64,65]. The relationship between PN and gs further illustrates the functional importance of water availability to photosynthetic processes. While significantly correlated in both seasons, the variables were more strongly correlated in the dry season than in the wet season. We show that the variation in gs when rain was excluded, was strongly related to VPD (r ¼
0.935; p  0.001), and in turn, VPD was strongly related to PN (r ¼
0.903; p  0.001). The weak relationship in the data collected during the rainy season, which has a narrower VPD range than during the dry season, may explain why previous studies carried out on J. curcas leaves [8,66] in environments with a narrow ranges in VPD conditions have concluded that VPD is not explanatory of gs or PN. Our findings extend these prior studies, showing that VPD has a larger effect than soil water on both gs and PN. The stomatal closure at high VPD observed during this study agrees with the results from other studies of J. curcas [7,8,67,68]. This species has an apparent ability to exploit the soil water deficit, responding rapidly to extremely high evaporative demand. This conclusion is represented in this study by the VPD, which reached values > 3 kPa during the dry season and 2.7 and 1.9 kPa during the wet season in the dry tropical and semi-humid tropical plants, respectively. Using CCA, Batista, Araújo [69] demonstrated that VPD is a significant climatic variable, explaining at least 65% of the total variance of PN. Similar behavior was found in field-grown plants, such as coffee [69], papaya [70], umbu [71] and J. curcas [7,8,66,68]. All these studies attributed the reduction in PN and gs to high leaf-to-air vapor pressure deficit, leading to stomatal closure. It is also worth noting that at low or very low gs, the Ci:Ca ratio was higher than it was expected (0.65e0.75) suggesting that the internal concentration of CO2 was not limiting for photosynthesis in J. curcas (Fig. 6D). This may be ascribed to carbon uptake limitation imposed by non-stomatal factors. Among possible causes for this limitation is a decrease in carboxylation efficiency caused by direct inhibition of the photosynthetic apparatus [72]. However, studies have reported that, in some cases, carboxylation efficiency decreases while gs remains constant [73]. Independent of gas exchange, leaves from dry ecosystems,

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compared with their semi-humid counterparts, had more abnormal stomata. The reasons for altered stomatal morphology are not yet understood. Recent studies have suggested that stomatal malfunctioning in low RH-grown leaves is a consequence of a high foliar abscisic acid content during leaf development at decreased atmospheric humidity, compared with leaves grown in a more humid atmosphere [21,74]. However, it has been shown that shorter stomata have faster response times than larger ones [24]. They could, therefore, open at sunrise and close more quickly, allowing CO2 fixation at times of the day that are associated with low transpiration rates (low VPD), resulting in higher WUEi [22,24,75]. We have shown that the strong correlations support the idea that high plasticity in SD would be closely associated with PN and WUEi in J. curcas leaves [1,13]; smaller stomata require less severe leaf drying to close [17], contributing to increased WUEi. Thus, water-limited environments are expected have species with the greatest WUEi [1,13]. An increase in WUEi with high SD might also indicate a high capacity to acclimate to a gradually increasing water deficit and suggests an evolutionary adaptation to environmental stresses in J. curcas [76e78]. 5. Conclusions The capacity to adjust leaf traits in response to seasonal variation makes J. curcas a good choice for energy crop cultivation. However, gs and PN are more likely to be limited by water availability in plants grown in semiarid areas than in plants grown in the semi-humid regions. Additionally, the decline in soil water content during the dry season in the semi-humid region does not seem to be large enough to reduce the plant's PN. Our results suggest that J. curcas may also contribute mitigating the effect of CO2 deposition in the atmosphere, given that a remarkable change in SD and other leaf traits was observed in response to seasonal variations. Finally, this specie has strong potential for acclimating to the altered environment predicted for the near future in the semiarid regions of Caatinga. Moreover, multivariate analysis highlights the high sensitivity of J. curcas plants to DPV which in turn induces rapid stomatal closure and consequent reduction of PN with possible fall in Ci for long periods of time which reflect into a change in the pattern of development resulting in higher SI. It is believed that in turn, via feedback that it minimizes the effect of low gs with increased stomatal frequency and facilitating the flow of CO2 into the leaf in a dry condition and whose diffusibility of CO2 is reduced. Acknowledgements The authors would like to thank the National Council for Scientific and Technological Development, CNPq (Grants 470476/ 2011-7) and Foundation for Science and Technology of Pernambuco (Grants APQ-0150-2.03/08) for financial support. Special thanks to Dr. Marcelo Guerra, Laboratory of Plant Cytogenetics and Evolution for logistical support in the utilization of the microscope and image processing. References [1] B. Singh, K. Singh, G. Shukla, V.L. Goel, U.V. Pathre, T.S. Rahi, et al., The field performance of some accessions of Jatropha curcas L. (biodiesel plant) on degraded sodic land in North India, Int. J. Green Energy 10 (10) (2013) 1026e1040. [2] M.F. Pompelli, D.T.R.G. Ferreira, P.P.G.S. Cavalcante, T.L. Salvador, B.S. Hsie, L. Endres, Environmental influence on the physico-chemical and physiological properties of Jatropha curcas L. seeds, Aust. J. Bot. 58 (6) (2010) 421e427. [3] N. Sunil, V. Kumar, M. Sujatha, G.R. Rao, K.S. Varaprasad, Minimal descriptors for characterization and evaluation of Jatropha curcas L. germplasm for utilization in crop improvement, Biomass Bioenerg. 48 (1) (2013) 239e249.

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