Effects of seed storage time and salt stress on the germination of Jatropha curcas L.

Industrial Crops & Products 118 (2018) 214–224

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Effects of seed storage time and salt stress on the germination of Jatropha curcas L.

T

Flavio Lozano-Islaa, Mariana L.O. Camposa, Laurício Endresb, Egídio Bezerra-Netoc, ⁎ Marcelo F. Pompellia, a

Plant Physiology Laboratory, Federal University of Pernambuco, Department of Botany, Recife, PE 50670901, Brazil Plant Physiology Laboratory, Federal University of Alagoas, Maceió, AL, Brazil c Department of Chemistry, Federal Rural University of Pernambuco, Recife, PE, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Salt tolerance Seed storage Seed germination Biofuel Carbon credit NaCl

Jatropha curcas L. has been proposed to be a potential tropical plant for augmenting renewable energy sources. Due to its several merits, J. curcas deserves to be considered as candidate for biofuel production instead of for food seed sources. Its seeds contain 27–40% oil that can be processed to produce a high quality biodiesel fuel that is usable in a standard diesel engine. While J. curcas grows well under low-rainfall conditions, the salt tolerance capacity of this species has received meager attention. Furthermore, another major problem faced by this species is the lack of a safe system capable of guaranteeing seed germination when stored for medium or long periods of time. Thus, J. curcas seeds show two important problems: (i) rapid loss of viability, resulting from the high respiratory rate of the seeds during storage, and (ii) seed sensitivity when germinated under salt conditions. Therefore, the main objectives were (i) to verify if storage in very low humidity boxes can improve germination without affecting the oil content of the seeds and (ii) to compare different genotypes to prove their salt tolerance capacities. The results indicate that from the use of desiccants, all the germination parameters were kept reasonably constant, and germination was essentially unchanged during the entire storage period. Biochemical activity, supported by the drastic reduction in respiratory activity of the seeds, was also decreased during the storage in order to keep the seed reserves intact; therefore, J. curcas seeds may be resistant to storage when stored in a controlled process. On the other hand, it was shown that J. curcas presents a moderate tolerance to salinity. This species is able to germinate in the presence of up to 150 mM of Sodium chloride (NaCl), although a drastic reduction in the biomass accumulation was observed with the increase in salt concentration in the irrigation water. It was shown that the germination was reduced to values close to 4% at 150 mM NaCl; whereas biometric and biomass characteristics were strongly affected by the increase in salt content. However, it was shown that genotypes 114, 171 and 183 were shown to be potentially tolerant, whereas genotypes 218 and 133 were sensitive.

1. Introduction There is a growing consensus that the use of the dirtiest fossil fuels may have leveled off (Horan, 2017). At the same time, renewables, particularly wind and solar, are booming. For example, demand for oil and gas is growing, and most electricity still comes from fossil fuels. Aviation and shipping have no viable alternative to hydrocarbons. However, several reports have shown that the replacement of kerosene used in aviation can be, at least, in part replaced by biofuels (Airbus, 2016; Baroutian et al., 2013; Gutiérrez-Antonio et al., 2016; Jim, 2009). In addition, the impending shortage of fossil fuels has caused humanity to rationally use the primary energy sources, consequently, new

⁎

Corresponding author. E-mail address: [email protected] (M.F. Pompelli).

https://doi.org/10.1016/j.indcrop.2018.03.052 Received 22 July 2017; Received in revised form 21 March 2018; Accepted 23 March 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

technologically developed versions of power plants have been designed in order to increase not only energy efficiency but also ecological efficiency. From these concepts, the idea of carbon credits arose. Carbon credits create a market for reducing greenhouse gases emission by giving a monetary value to the cost of polluting the air. In this sense, Jatropha curcas (purging nut) comes into play, because this species requires little water and fertilizer (Achten et al., 2010; Pompelli et al., 2010a; Sapeta et al., 2013), can survive on infertile soils and is not grazed by cattle (Sarin et al., 2007), making this species suitable for cultivation on degraded soils (Achten et al., 2010). Furthermore, J. curcas should be the opportunity to gain future project financing through carbon credits (Horan, 2017; Torres et al., 2011; van Rooijen,

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distinct genotypes of J. curcas exposed to different NaCl treatments were studied to determine their tolerance and to understand the morphological and physiological responses of these species under conditions of salinity with respect to germination and initial development. Thus, the main hypotheses of this work were (i) to verify if the use of a desiccant agent could help maintain the viability and germinability of the seeds of J. curcas when stored for long periods of time and (ii) to study the mechanism of the tolerance of J. curcas to salinity among the some genotypes cultivated in Brazil.

2014; Wani et al., 2012). The majority of the present projects combine plantations and small-scale production (Divakara et al., 2010; Pandey et al., 2012). J. curcas is a species belonging to the Euphorbiaceae family and has multiple uses; this species is widely distributed in many tropical and subtropical regions in the Americas, Africa and Asia (Achten et al., 2010). Over the past 20 years, this species has gained much attention as a potential crop for bioenergy production, since its seed oil can easily be converted to high-quality biodiesel. In addition, this species is not edible and therefore does not compete with the other oilseeds used as food sources, such as maize and soy (Pompelli et al., 2011). J. curcas is easily propagated and has a growth rate, short period until the first fruit harvest, low seed cost (Heller, 1996), high oil content (40–58%) (Pandey et al., 2012; Pompelli et al., 2010b), and good adaptation to different agroclimatic conditions (Divakara et al., 2010; Fini et al., 2013). In taking into account that J. curcas is a potential species for the large-scale generation of biodiesel, it is easy to think that it will be necessary to plant hundreds or thousands of hectares of trees to produce a satisfactory amounts for commercial exploitation (Contran et al., 2013). In this sense, it is also important to remember that in times of harvest, market prices usually fall sharply (Summer and Mueller, 1989), which is where the storage of seeds comes into play. Seed storage is one the most important factors that negatively affect seed viability, which includes the time elapsed between harvest and utilization (MarcosFilho, 1998; Marcos-Filho et al., 1984). Marcos-Filho (1998) reported that seed storage is a major problem for agriculture, as seed storage is responsible for considerable losses throughout the world, especially in the tropics (Tekrony, 2006), where high temperatures and high relative humidity prevail during seed maturation and storage (Marcos-Filho, 1998). J. curcas does not escape this pattern, as it presents high metabolism, causing its seeds to rapidly lose their viability during storage (Moncaleano-Escandon et al., 2013). Although deterioration is irreversible and unavoidable, the speed of the process can be controlled by appropriate harvest, drying and storage techniques (Summer and Mueller, 1989). In this sense, the use of a drier atmosphere can protect seeds (Hay et al., 2012; Rao et al., 2006), and slow down the respiratory process, which is responsible for the loss of organic compounds that will be used as energy sources by the embryo during germination (Chidananda et al., 2014). Another factor that negatively influences agriculture, mainly in irrigated crops, is salinity (Kumar and Sharma, 2008), with NaCl being the predominant salt. At the world level, approximately 33% of agricultural lands irrigated by saline water are facing the problem of salt stress (Shrivastava and Kumar, 2015). Seed germination begins with imbibition of the quiescent seed and ends with the elongation of the embryonic axis, at which point the reserves begin to be mobilised to provide energy to the developing embryo. Salinity can also affect germination by limiting the absorption of water in the seeds (osmotic effect) (Almansouri et al., 2001; Hegarty, 1977), increasing the toxicity of ions or the combination of both (Apse et al., 1999). In addition, NaCl can affect the mobilization of reserves (Bouaziz and Hicks, 1990), structural organization and protein synthesis in embryos (Alencar et al., 2015). In saline environments, plant adaptation during germination involves decisive stages for species establishment, and such factors can negatively influence the germination process (Ungar, 1995). Furthermore, salinity affects plant growth and development (Munns and Tester, 2008), negatively influencing the stages of its development (Almansouri et al., 2001; Kumar and Sharma, 2008). However, throughout their evolution, plants have developed mechanisms for regulation and tolerance to salts. Some studies have described the ecophysiological aspects of the tolerance of J. curcas to NaCl (Campos et al., 2012; Díaz-López et al., 2012; Rajaona et al., 2012); however, these studies focused on only one genotype, and there is no known research that has examined the effect of NaCl on different genotypes during germination and early seedling growth. In this study, five

2. Materials and methods 2.1. Aging tests To test the effect of storage on seed viability, an artificial aging test was used to reduce the water content in the interstices of the seeds with a desiccant material composed of silica gel (Sigma-Aldrish, part number 10087, Sigma-Aldrich Chemie, Schnelldorf, Germany). The genotype used in this experiment was 171 from Maceió, AL, Brazil. In each experimental unit, 50 seeds of J. curcas were arranged in germination boxes (110 mm × 110 mm × 35 mm) were added under a stainless steel mesh suspended 2 cm from the desiccant. In each germination box, 1.5 g of silica gel were added per gram of seed (as previously tested where it was shown to be the best in preventing vigor, and seed germination; Supplementary Fig. 1). Four storage times (i.e., 0, 3, 6, 9, and 12 months, with zero representing non-stored seeds) were tested and stored in a refrigerator at 4 ± 2 °C. After each storage time, the seeds were removed from their storage boxes and then allowed to germinate.

2.2. Germination tests with aged seeds After removal from the germination boxes that contained the desiccant, the seeds were allowed to germinate in other germination boxes (110 × 110 × 35 mm) that contained two sheets of germination test paper soaked with twice the weight of the paper in water; the boxes were sealed with Parafilm® M (Sigma-Aldrish, part number P7793-1EA, Sigma-Aldrich Chemie, Schnelldorf, Germany) and placed in a growth chamber (mod. NT 708, New Technical Instruments, Piracicaba, SP, Brazil). The incubator was equipped with four cold white fluorescent lamps of 20 W that produced 40 μmoles m−2 s−1 at the level of the germination boxes. The photoperiod was 12 h, and the temperature conditions were 25 ± 0.5 °C. Germination was evaluated daily for a period of 25 days. The seeds whose radicles had emerged from the integuments were considered germinated.

2.3. Biochemical analysis of the seeds used in the aging test A portion of 30% of the aged seeds was carefully ground in liquid nitrogen and stored at −20 °C until use. For extraction of soluble carbohydrates (TSC), soluble amino acids (TSA) and starch (STR), samples were solubilized in 50% (v/v) ethanol (Trethewey et al., 1998), whereas for the analysis of total soluble proteins (TSP), the samples were extracted in Stitt buffer (Armengaud et al., 2009). To measure soluble carbohydrates and starch, soluble proteins and soluble amino acids, the methods described by Dubois et al. (1956), Bradford (1976) and Moore and Stein (1954), respectively, were used. For the quantification of the oil content (OIL), the method described in detail by Ahmad et al. (1981) was used. To quantify the glucose (GLC), fructose (FTS) and sucrose (SCR), coupled to the production of 6-phosphogluconate, in the sequential presence of hexokinase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and invertase enzymes was used, as described by Stiit et al. (1989). All these analyzes were performed in triplicate. 215

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2.6. Evaluation of biometric parameters and biomass in salt experiments

Table 1 Information and location of the five genotypes of Jatropha curcas studied under salinity conditions. Genotype

City

State

Location

Altitude (masl)

183

Jaíba

Minas Gerais

478

114

Umuarama

Paraná

218

Goiás

171

São Miguel do Araguaia Maceió

133

Santa Inês

Maranhão

23°47′55.0”S 53°18′48”W 15°10′27.0”S 43°53′18”W 13°55′57.0”S 50°09′17”O 09°27′60.0”S 35°49′41”W 03°39′24.9”S 45°22′36”W

Alagoas

Mean seedling height (HGT) and mean stem diameter (STD) were evaluated at the end of the experiment. For this, the seedlings were collected and separated into three components: leaves, stems and roots. The stem diameter was measured using a digital caliper (Digital Caliper, ROHS, ZAAS Precision, Piracicaba, SP). Leaf area (LFA) was evaluated per plant and per experimental unit (TLFA) (Pompelli et al., 2012). To measure dry biomass, all samples were dried in a forced-ventilation oven at 70 °C for 72-h. Leaf dry weight (LDW), stem dry weight (STDW) and root dry weight (RDW) were used to calculate the biomass parameters: total dry weight (TDW); Leaf weight ratio (LWR; ratio between LDW and TDW); stem dry weight ratio (SWR; ratio between STDW and TDW); shoot dry weight ratio (STWR, ratio between STDW and TDW) and root dry weight ratio (RWR; ratio between RDW and TDW).

430 350 131 31

2.4. Physiological analyses coupled to the aging test

2.7. Evaluation of germination parameters

The relative water content (RWC) of seeds was calculated according to the method of Moncaleano-Escandon et al. (2013). The water potential (WTP) of the seeds was quantified using a dewpoint water potential meter (WP4C; Decagon Devices, Pullman, WA, USA); the seeds were lightly cracked to allow water to pass through the seeds to their internal environment. The values were obtained in MPa. For respiratory rate estimation (RPR), 10% of the seeds used at each storage time were inserted whole in a CO2 flow chamber (6400-09; LiCOR, Lincoln, NE, USA). For each storage time, 10 different samples were used as replicates. In each measurement procedure, three cycles of 102-s were performed, with a 2-s interval between the readings. During this time, the increase in CO2 concentration inside the chamber was monitored. The reference CO2 was calibrated at each measurement according to the CO2 concentration of the environment (∼ 400 μmol CO2 m−2 s−1). The net respiration rate was expressed in μmol CO2 h−1 g−1 seed.

For both experiments, the different germination parameters were calculated: germinability (GRP), mean germination time (MGT), germination uncertainty (GRU) and germination synchrony (GRS). All calculations were performed with GerminaR package (Lozano-Isla et al., 2017).

2.5. Germination tests in the presence of NaCl

3. Results

2.8. Experimental design and statistical analysis Statistical analysis and the generation of graphs were performed using the statistical software R (de Mendiburu, 2016). The analysis of variance (ANOVA) was performed to evaluate the differences between the factors, and the means compared using the Student-Newman-Keuls test (p < 0.05) (de Mendiburu, 2016). For the multivariate analysis, correlation analysis was performed (de Mendiburu, 2016; Wei and Simko, 2016), and principal components analysis was performed as recommended by Husson et al. (2017).

For this experiment, the seeds of five J. curcas genotypes (Table 1) originating from different producing regions of Brazil, given by Tropical Semiarid Agricultural Research Center (Embrapa Agroenergy; Brasília, DF − Brazil), were kept at 4 °C until their use (Moncaleano-Escandon et al., 2013). Germination was carried out in a greenhouse at the Federal University of Pernambuco, Department of Botany, Recife, PE (8°02″59.0′S; 34°56″54.9′W, 4 masl). The average temperature was recorded during the experiments using a portable mini climate station (mod. KR420, Akron Measure Instrument, Leuven, Belgium) every fifteen minutes, 24 h per day. The mean temperature and relative humidity along the germination were, respectively, 30.6 ± 1.1 °C and 70.4 ± 5.8%. In this experiment, five different concentrations of NaCl (0, 50, 75, 100 and 150 mM) were tested in the irrigation water; the zero concentration was distilled water only. After soaking, all seeds were disinfected with NaOCl (2%) for 10-min and triple rinsed with distilled water. Afterward, the seeds were germinated in polypropylene boxes (200 mm × 200 mm × 50 mm) containing 2500 g of washed river and were air-dried. Each box containing 25 seeds was considered an experimental unit. The boxes were irrigated daily with 300 mL of water containing Hoagland nutrient solution (Epstein, 1972) at a concentration of 25%, this amount was the volume of irrigation required (i.e., previously tested) to leach excess salts and prevent their accumulation in the media. Germination was evaluated daily for 25 consecutive days until the seedlings were collected. In this experiment, seed was considered germinated when the aerial part emerged from the soil. The experiment were replicated five times.

3.1. Germination of aged seeds The germination of J. curcas seeds subjected to storage ranged from 9% to 15%, and the values were statistically similar to each other (Fig. 1A). Although germination was not affected by storage time, the mean germination time was significantly increased with storage time (Fig. 1B). Germination was completely asynchronous for all storage times, with a mean of 0.16 recorded before storage and a mean close to zero recorded at other times (Fig. 1C). Therefore, germination uncertainty was not affected by storage (Fig. 1D). It was verified that the seeds before storage and seeds stored for 3 months began their germination on the 3rd and the 4th day after sowing, respectively; thereafter, germination of the first seed was computed only after the 6th day (Fig. 2). The time until the stabilization of germination increased as the storage time was increased and was completed after 15 days in nonstored seeds and after 23 days in seeds under 12 months of storage. 3.2. Biochemical responses of seeds subjected to aging It was verified that the oil content in the seeds remained essentially stable until the 6th month of storage at a rate of 35%, with slight reductions occurring until the 12th month of storage when the oil content was approximately 29% (Fig. 3A). It was verified a strong and negative correlation between oil content in the seeds and storage time (r = −0.91, p ≤ 0.05). On the other hand, starch was rapidly metabolized with an approximate reduction of 33% after 12 months of storage (Fig. 3B). The total soluble protein and total soluble amino acid contents increased by 160% and approximately 67% during storage, respectively (Fig. 3C–D). A strong and positive correlation (r = 0.92, 216

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Fig. 2. Cumulative germination during the time evaluated in Jatropha curcas after storage for 0, 3, 6, 9, and 12 months.

p ≤ 0.05) was shown between total soluble protein content and amino acid synthesis. There was a gradual reduction in the total soluble carbohydrate content after the 3rd month of storage compared with that of non-stored seeds, but differences were not observed until the 12th month of storage (Fig. 4A). Sucrose levels decreased by approximately 49% between 3 and 12 months of storage (Fig. 4B), while glucose levels remained stable until the 3rd month of storage compared to those of non-stored seeds; however, from the 6th month, the glucose was rapidly increased and reached 71% in the 12th month in relation to that of nonstored seeds (Fig. 4C). On the other hand, fructose levels did not show a trend with time (Fig. 4D). It was shown that while sucrose levels decreased (r = −0.57, p ≤ 0.05) throughout the storage time, glucose exhibited the opposite behavior (r = 0.67, p ≤ 0.05) and increased with storage time. 3.3. Physiological responses of seeds subjected to aging With increasing storage time of the desiccant agent, it was verified that the water content in the seeds was steadily decreased. Non-stored seeds had 8% of water content, while seeds stored after 12 months had 5.5% (Fig. 5A). Concomitantly, the seed water content and water potential severely decreased with the storage time, and a strong correlation (r = 0.83, p ≤ 0.05) was present between these two features. It was verified that the water potential of non-stored seeds was −35 MPa, but water potential decreased to −124 MPa at 12 months of storage (Fig. 5B). A reduction in the relative water content and the water potential, led to a strong reduction in the respiratory rate of the seeds (r = 0.88, p ≤ 0.05), ranging 115 mmol CO2 h−1 g−1 MF in non-stored seeds to 10 mmol CO2 h−1 g−1 MF in seeds after 12 months of storage (Fig. 4). A reduction of 91% in favor of non-stored seeds occurred (Fig. 5C). 3.4. Seed germination under NaCl All germination parameters showed significant effects of salt concentration and an interaction between salt and genotype, i.e., the effect of salt stress was not similar in all genotypes. In this experiment, it was verified that the germination was almost zero at 150 mM NaCl for all genotypes. At 0 mM NaCl, the seeds of genotypes 183 and 114 exhibited 71% and 86% germination, respectively, and gradually decreased with the increasing NaCl concentration, from 4% at 100 mM NaCl and 0% at 150 mM NaCl (Fig. 6A). In another way, genotypes 218 and 133 did not differ up to 100 mM NaCl, although germination was reduced to approximately 5% at the concentration of 150 mM NaCl. The seeds of genotype 171 exhibited 65% germination in the control, which

Fig. 1. Germinability (A), mean germination time (B), synchrony index (C), and germination uncertainty (D) evaluated in Jatropha curcas seeds after storage for 0, 3, 6, 9, and 12 months. The bars represent the means ( ± SE). The mean differences between storage months are represented by the lowercase letters (SNK, p = 0.05). n = 4.

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Fig. 3. Oil content (A), starch (B), total soluble protein (C), and total soluble amino acids (D) evaluated in Jatropha curcas seeds after storage for 0, 3, 6, 9, and 12 months. The bars represent the mean ( ± SE). The mean differences between the storage months are represented by the lowercase letters (SNK, p = 0.05). n = 4.

Fig. 4. Total soluble carbohydrates (A), sucrose (B), glucose (C), and fructose (D) evaluated in Jatropha curcas seeds after storage for 0, 3, 6, 9, and 12 months. The bars represent the mean ( ± SE). The mean differences between the storage months are represented by the lowercase letters (SNK, p = 0.05). n = 4.

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Fig. 5. Relative water content (A), water potential (B), and seed respiratory rate (C) evaluated in Jatropha curcas after storage for 0, 3, 6, 9, and 12 months. The bars represent the mean ( ± SE). The mean differences between the storage months are represented by the lowercase letters (SNK,p = 0.05). n = 4.

decreased to 32% and 9% at 50 mM NaCl and 150 mM NaCl, respectively (Fig. 6A). The mean germination time under 0 mM NaCl was 5–7 days for all genotypes, while under 150 mM NaCl the interval was longer, ranging from 7.2 days to 12.3 days. No significant differences were observed for mean germination time from 50 mM NaCl to 75 mM NaCl, and the general average was 7.5-days for all genotypes (Fig. 6B). There was no difference in germination synchrony for the genotypes up to 75 mM NaCl. However, synchronization at 100 mM NaCl was null for genotypes 183, 114 and 218, as well as for genotype 133 at the concentration of 150 mM NaCl (Fig. 6C). It was observed that the synchrony was always less than 0.25 for all concentrations and genotypes (Fig. 5C), denoting an asynchronous profile. The maximum value for the uncertainty of germination in this experiment was 4.64 bits. Genotypes 183 and 114 showed a tendency to reduce uncertainty with an increase in NaCl concentration. The germination uncertainty was stable up to 75 mM NaCl, but significantly increased at the concentration of

Fig. 6. Germinability (A), mean germination time (B), germination synchrony (C), and germination uncertainty (D) evaluated in five genotypes of Jatropha curcas under different NaCl concentrations (0, 50, 75, 100 and 150 mM). The vertical bars represent the means ( ± SE). The mean differences between the accessions are represented by different capital letters and between salt levels by different lowercase letters (SNK, p = 0.05). n = 4. 219

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100 mM NaCl, where the uncertainty values were 0.5 and 0.3 for genotypes 183 and 114, respectively (Fig. 6D). Genotypes 218, 171 and 133 showed a trend in increasing uncertainty from 0 mM NaCl to 100 mM NaCl without showing significant differences. At 150 mM NaCl, the uncertainty values were 0.3, 0.7, and 0.5 for genotypes 218, 171 and 133, respectively (Fig. 6D). Germination over time showed differences for each of the genotypes. Germination at 0 mM NaCl began between the 3rd and 4th day after sowing, whereas for the treatments with NaCl addition, the maximum values of germination were observed between the 9th and 12th days (Fig. 7A–E). Regardless of the genotype, first germination was observed on the 3rd day at 0 mM NaCl, while at 50 mM NaCl and 75 mM NaCl, the germination was generally initiated on the 4th day; germination began on the 5th day and 7th day at 100 mM NaCl and 150 mM NaCl, respectively. However, regardless of treatments and salinity levels, germination became stable after the 13th day (Fig. 7). 3.5. Biometric and biomass components Although germination was evaluated to a concentration of 150 mM NaCl, the biomass production was computed only up to 100 mM NaCl because above this concentration the plants did not have enough vigor to resist salinity and eventually languished. Similar to seed germination, all plant growth parameters and ratios responded differently to salt stress, which can be seen by the significant interaction among factors for almost all biometric variables (Table 2). There was a decreasing trend regarding plant height (HGT) with increasing salt stress (Fig. 8A). The HGT reached approximately 13 cm at 0 mM NaCl but was strongly reduced to less than 3 cm at 100 mM of NaCl, a fact clearly recorded for genotype 183. The root dry weight ratio (RWR; Fig. 8B) showed a distinct behavior for each genotype, with genotypes 171, 133 and 218 increasing their root biomass under conditions up to 75 mM NaCl. On the other hand, the genotype 183 showed increases under conditions up to 50 mM NaCl but its values decreased thereafter. Genotype 114 was seemingly unaffected by the increase in NaCl concentration (Fig. 8B). Stem diameter (STD) presented a reduction with the increase in the concentration of NaCl, despite no significant differences for concentration from 50 to 100 mM NaCl for genotypes 218, 171 and 133. At concentrations of 100 mM NaCl, the genotype 183 had the lowest values of STD (Fig. 8C). A distinct profile was verified in the stem dry weight ratio (SWR), which increased with increasing salinity, even though no significant effect was observed up to the 50 mM NaCl concentration. For the 75 mM NaCl concentration, genotype 183 performed better than the others did, while the genotypes 183, 171 and 133 showed the best SWR at 100 mM NaCl (Fig. 8D). The leaf area (LFA) could be evaluated until concentration of 75 mM NaCl, as leaf area was the most affected parameter by the increase of NaCl in the irrigation solution. Genotype 218 presented the highest values for 0 mM NaCl, while at 50 mM NaCl the genotypes 133, 114 and 183 showed the highest performance. For the leaf dry weight ratio (LWR), it was possible to observe a trend in the reduction in biomass according to increasing salinity, with genotype 218 presenting a ratio of 0.42, followed by genotype 183 with a ratio of 0.39 at 0 mM NaCl (Fig. 8F). Genotype 171 did not show significant differences in leaf weight ratio up to 50 mM NaCl, while genotype 114 increased its biomass accumulation at concentrations up to 50 mM NaCl. All genotypes showed a drastic reduction of LWR beginning at 75 mM NaCl (Fig. 8F). 3.6. Multivariate analysis of the salinity experiment Fig. 7. Cumulative germination during the time evaluated in five genotypes of Jatropha curcas under different NaCl concentrations (0, 50, 75, 100 and 150 mM). Genotype 183 (A), 114 (B) 218 (C), 171 (D), and 133 (E).

Germination parameters were negatively affected by the increase in the salt concentration. There was a negative correlation between salt increase and both germination percentage (r = −0.55, p ≤ 0.05) and germination synchrony (r = −0.69, p ≤ 0.001). On the other hand, the mean germination time was positively significant when correlated with 220

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Table 2 Summary of the analysis of variance of the seed germination and seedling growth parameters evaluated in five genotypes of Jatropha curcas under different NaCl concentrations (0, 50, 75, 100 and 150 mM). SV: Source of variation; DF: degrees of freedom; GRP: Germination percentage; MGT: mean germination time; SYN: germination synchrony; UNC: germination synchrony; HGT: plant heigth; STD: stem diameter; LFA: leaf área; LWR: leaf weigth ratio; RWR; root weigth ratio; STWR: shoot weigth ratio; SWR: stem weigth ratio. *Singnificant at p < 0.05 by F-test. SV Genotype Salt (Genotype x Salt) Error

DF 4 4 16 75

GRP

MGT *

2527.04 6662.64* 1249.54* 61.6

SYN *

15.775 43.453* 2.947* 1.228

UNC *

0.023 0.052* 0.013* 0.005

HGT *

1.697 10.521* 1.184* 0.25

STD *

46.919 1088.695* 7.454* 2.02

salinity (r = 0.66, p ≤ 0.001). However, there was no significant correlation between germination uncertainty and salt increase (p = 0.321). The salt concentration negatively affected all biomass parameters, e.g., r = −0.86 (to leaf dry weight), r = −0.89 (to leaf area), r = −0.89 (to plant height) and r = −0.91 (to stem diameter). In a similar manner, all growth ratios were negatively influenced by salt, e.g., r = −0.90 (to leaf weight ratio), r = −0.94 (to leaf area ratio). However, stem dry weight ratio was positively influenced

LFA *

2.855 90.008* 4.02* 0.56

LWR *

719.304 51524.82* 1240.02* 51.738

*

0.02 0.467* 0.011* 0.004

RWR

STWR *

0.079 0.062* 0.034* 0.002

*

0.079 0.062* 0.034* 0.002

SWR 0.037* 0.562* 0.042* 0.007

(r = 0.74) by salt addition. The principal components analysis (PCA) showed that approximately 74.4% of the variation can be explained by the parameters studied here. In the first component, the variables LDW, LFA, HGT and TDW were the ones that presented the greatest contribution to variance and were negatively correlated with the increase in NaCl, while the variables with positive correlation included MGT and SWR. Genotypes 114, 171 and 183 were the best among all tested genotypes, while 133 and 218 seemingly were salt susceptible (Fig. 9).

Fig. 8. Height (A), root dry weight ratio (B), stem diameter (C), stem dry weight ratio (D), leaf area (E) and leaf dry weight ratio (F) evaluated in five genotypes of Jatropha curcas under different NaCl concentrations (0, 50, 75, 100 and 150 mM). The vertical bars represent the means ( ± SE). The mean differences between the accessions are represented by different capital letters and between salt levels by different lowercase letters (SNK, p = 0.05). n = 4. 221

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Fig. 9. Principal component analysis (PCA) of the variables evaluated in five genotypes of Jatropha curcas under different NaCl concentrations (0, 50, 75, 100 and 150 mM). GRM, Germinability; MGT, mean germination time; UNC, Uncertainty of germination; SYN, Synchronism of germination; LDW, leaf dry weight; RDW, root dry weight; SDW, stem dry weight; TDW, total dry weight; LFA, leaf area; TLFA, total leaf area; LWR, leaf dry weight ratio; SWR, stem dry weight ratio; RWR, root dry weight ratio; STWR, shoot dry weight ratio; HGT, plant height; STD, stem diameter.

4. Discussion

seed moisture. Similarly, we showed a significant correlation between the decrease in sucrose and increase in both glucose (r = −0.87, p ≤ 0.05) and fructose (r = −0.56, p ≤ 0.05). The total soluble carbohydrate content must have been reduced by the metabolism of sucrose, which acted as a carbon source for the embryo. Circumstantial evidence of this idea is based on the elevation of glucose and fructose contents, while there was a reduction in the levels of starch and sucrose. Thus, the respiration rate, despite being low, enabled the embryo to live. This phenomenon resulted in the same germination rate, even after 12 months storage − a fact that diverges from the report of Moncaleano-Escandon et al. (2013). These authors reported that the germination of J. curcas seeds drop to near to zero after 12 months of storage; however, six months after the start of the experiment, the seed germination decreased by 31% compared to that of non-stored seeds. Additional strong evidence regarding respiration being responsible for the mobilization of reserves can be corroborated by the strong negative correlations between oil, starch and sucrose contents and their degradation products (i.e., total soluble proteins, total soluble amino acids and glucose). The respiration rate also increased with seed moisture content, and seed respiration increased linearly with storage (Chidananda et al., 2014). In their studies, Moncaleano-Escandon et al. (2013) and Chidananda et al. (2014) did not use any desiccant to prevent or decrease respiration rates in stored seeds. In addition, it was reported that seeds containing between 6% and 8% of moisture

The seeds of J. curcas are classified as orthodox (Hay et al., 2012). They are resistant to desiccation and can present water contents up to 18% (Pompelli et al., 2010b) when freshly harvested. MoncaleanoEscandon et al. (2013), reported that the seeds of J. curcas can drastically exhibit reduced germinability during storage at temperatures of 4 °C or 25 °C, accompanied by the reduction of some biochemical compounds such as starch and total soluble proteins. MoncaleanoEscandon et al. (2013) also reported that at 4 °C the results were more interesting regarding the seed storage of J. curcas. Thus, all our tests were performed at 4 °C as recommended by Moncaleano-Escandon et al. (2013). However, with the use of the desiccant, it was shown a reduction in relative humidity in the interstice of the seeds, reducing the water content of the seeds and, consequently, the osmotic water potential. Coupled with these facts, the respiration rate was strongly reduced but was not zero, which allowed the viability of the embryo at the expense of the solubilization of reserves; this phenomenon, in turn, supported the germinability of the seeds during storage without significant reductions. However, it was shown that a strong and positive correlation between total soluble proteins and amino acid synthesis. A possible explanation could be that structural proteins were mobilized to generate carbon skeletons for respiration or amino acids as compatible solutes, allowing maintenance of respiration even with a reduction in

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exhibited a sudden reduction in seed oil content in the first 3 months of storage together with an increase in the concentration of free fatty acids (Akowuah et al., 2012; Dharmaputra et al., 2009). Thus, it was verified that moisture control in the interstice of the seeds should be taken into account to preserve both the viability and the oil content of the seeds of J. curcas. The negative effects of saline stress were reflected by the delay in mean germination time, from 3 days at 0 mM NaCl to 13 days at 150 mM NaCl, which is confirmed by the significant reduction in germination rate. The same pattern was reported by Alencar et al. (2015), who described that J. curcas presents a strong and negative correlation between germination rate and mean germination time. Regardless of storage time, germination was initiated between the 3rd and 5th days, and was complete after 23 days of sowing, which occurred mainly after 12 months of storage. Both storage and salinity delayed germination. Among them, however, storage seems to be the factor that promotes better storage time, while NaCl seems to be toxic to the germination of J. curcas seeds. It was observed that the germination synchrony was reduced with the increase in salts concentration, and the salinity promoted a more disorganized germination − a fact corroborated by the high values of germination uncertainty. The fact that J. curcas exhibits asynchrony in germination, mainly in salt stress, is very well studied (Alencar et al., 2015; Islam et al., 2009; Moncaleano-Escandon et al., 2013; Pompelli et al., 2010b). Therefore, if we analyze that the published data together with those presented in this study, we can postulate that the synchrony and uncertainty of germination cannot be considered a good parameters for judgment, at least in J. curcas. A possible explanation for this could arise from the fact that J. curcas is not yet a domesticated species (Achten et al., 2010), which makes it present high levels of uncertainty in germination (Moncaleano-Escandon et al., 2013). This factor is strongly related to the survival of the species in its original habitat (Maes et al., 2009) and is very unstable from a physiological point of view. A delay in J. curcas seed germination accompanied by both a decrease in the development of the leaves and a reduction in the root growth promotes a delay in the autotrophic phase of the plants and, in extreme cases, leads to the death of seedlings in the first days after germination. A possible explanation for this phenomena was presented by Alencar et al. (2015), as there were strong increases in the Na+ and Cl− contents in the embryonic axes and in the endosperm of the seeds of J. curcas. Another possibility is the loss of the mobilization of cotyledon reserves during germination (Liu et al., 2010), which affects seedling establishment (Alencar et al., 2015). When subjected to salinity, plants often increase their stem biomass to the detriment of other organs (Díaz-López et al., 2012; Munns and Termaat, 1986; Praxedes et al., 2010). Munns and Termaat (1986) described this fact as an indirect effect of decreasing water uptake by roots and lower leaf expansion, while Praxedes et al. (2010) described this effect as a lower relative growth rate of the plant as a whole. Many studies (Bayuelo-Jiménez et al., 2002; Debez et al., 2004) have reported that all biometric components are reduced in non-halophyte plants when subjected to salinity. In addition, Alencar et al. (2015) attributed a number of factors to the negative effects of salt stress, such as changes in the water status of the plant caused by the osmotic effects of the salts and increases in the concentrations of toxic ions, which could lead to physiological and biochemical variations and to alterations to the absorption of essential nutrients, such as potassium and calcium. Taken together, these results indicate the following: (i) The viability of germination and oil content in the seeds of J. curcas can be maintained if there is control of moisture in the interstices of the seed during storage; (ii) Biometric parameters better explain the salinity response than germination parameters do; thus, genotypes 114, 171 and 183 would be considered interesting candidates for salt stress tolerance, whereas genotypes 133 and 218 showed sensitivity to NaCl addition; and (iii) Considering only salt stress experiments, it was concluded that genotypes, 114, 171 and 183 can be considered potential candidates for

future breeding programs. The goal is to allow market mechanisms to drive industrial and commercial processes in the direction of low emissions or less carbonintensive approaches than those used when there is no cost to emitting carbon dioxide and other greenhouse gases into the atmosphere. Because greenhouse gases mitigation projects generate credits, this approach can be used to finance carbon reduction schemes between trading partners and around the world (Johnson, 2009). In 2008, 123.4 million metric tones of carbon dioxide were transacted (Ingole et al., 2013), and the market was valued at US$ 705 million, with annual growth of 15% projected. India is considered to claim approximately 31% of the total world carbon trade, which can give 25 billions dollars by 2010 (Gupta, 2011). Therefore, J. curcas, if established in hot, dry, coastal areas around the world, could capture 17–25 tons of carbon dioxide per hectare per year from the atmosphere (over a 20-years period) (Becker et al., 2013). Therefore the planting of J. curcas can be exchanged for carbon credits, alleviating the effects of global climate change (Gupta, 2011; Ingole et al., 2013). In this sense, the planting of J. curcas could be used as an alternative source of clean and ecologically correct biofuel. 5. Acknowledgments The authors thank the National Council for Scientific and Technological Development (CNPq Process no 404357/2013-0) for the financial support. The first author thanks the Coordination of Improvement of Higher Education Personnel (CAPES) for the scholarship. Special thanks to Dr. Agnaldo Rodrigues de Melo Chaves, Tropical Semiarid Agricultural Research Center, Embrapa Semiárido, Petrolina, PE, Brazil and Embrapa Agroenergy, Brasília, DF, Brazil for giving the seeds used in this study. The corresponding author would also like to extend special thanks to Mrs. Ana Maria Meucci Dal Cin for her teaching and dedication to be better formation, approximately 30 years ago. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.indcrop.2018.03.052. References Achten, W.M.J., Nielsen, L.R., Aerts, R., Lengkeek, A.G., Kjær, E.D., Trabucco, A., Hansen, J.K., Maes, W.H., Graudal, L., Akinnifesi, F.K., Muys, B., 2010. Towards domestication of Jatropha curcas. Biofuels 1, 91–107. http://dx.doi.org/10.4155/bfs.09.4. Ahmad, M.U., Husain, S.K., Osman, S.M., 1981. Ricinoleic acid in Phyllanthus niruri seed oil. J. Am. Oil Chem. Soc. 58, 673–674. http://dx.doi.org/10.1007/bf02899445. Airbus, 2016. TAM Airlines and Airbus First to Fly Jatropha-based Biofuel in Latin America. Akowuah, J., Addo, A., Kemausuor, F., 2012. Influence of storage duration of Jatropha curcas seed on oil yield and free fatty acid content. ARPN J. Agric. Biol. Sci. 7, 41–45. Alencar, N.L.M., Gadelha, C.G., Gallão, M.I., Dolder, M.A.H., Prisco, J.T., Gomes-Filho, E., 2015. Ultrastructural and biochemical changes induced by salt stress in Jatropha curcas seeds during germination and seedling development. Funct. Plant Biol. 42, 865–874. http://dx.doi.org/10.1071/fp15019. Almansouri, M., Kinet, J.M., Lutts, S., 2001. Effect of salt and osmotic stresses on germination in durum wheat (Triticum durum desf.). Plant Soil 231, 243–254. http://dx. doi.org/10.1023/a:1010378409663. Apse, M.P., Aharon, G.S., Snedden, W.A., Blumwald, E., 1999. Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285, 1256–1258. http://dx.doi.org/10.1126/science.285.5431.1256. Armengaud, P., Sulpice, R., Miller, A.J., Stitt, M., Amtmann, A., Gibon, Y., 2009. Multilevel analysis of primary metabolism provides new insights into the role of potassium nutrition for glycolysis and nitrogen assimilation in Arabidopsis roots. Plant Physiol. 150, 772–785. http://dx.doi.org/10.1104/pp.108.133629. Baroutian, S., Aroua, M.K., Raman, A.A.A., Shafie, A., Ismail, R.A., Hamdan, H., 2013. Blended aviation biofuel from esterified Jatropha curcas and waste vegetable oils. J Taiwan Inst. Chem. Eng. 44, 911–916. Bayuelo-Jiménez, J.S., Craig, R., Lynch, J.P., 2002. Salinity tolerance of Phaseolus species during germination and early seedling growth. Crop Sci. 42, 1584–1594. http://dx. doi.org/10.2135/cropsci2002.1584. Becker, K., Wulfmeyer, V., Berger, T., Gebel, J., Münch, W., 2013. Carbon farming in hot, dry coastal areas: an option for climate change mitigation. Earth Syst. Dynam. 4,

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