Ruptura dormente e níveis de hormônios em sementes de Jatropha curcas l. e Jatropha macrocarpa griseb Dormant rupture and hormones levels in Jatropha curcas l. and Jatropha macrocarpa griseb seed

J. curcas L. and J. macrocarpa Griseb. are perennial shrub with the greatest importance mainly from its biofuel potential. Several authors consider that seed tegument is one of the factors that induce dormancy. The aim of the work was to study the role of tegument and abscisic acid (ABA) y jasmonic acid (JA) in dormancy and germination in these species . J. macrocarpa present dormancy since it does not germinate by traditional methods. Consequently, seeds of J. macrocarpa were subjected to different treatments to break seed dormancy: T1) Control; T2) Scarification with sandpaper; T3) Total elimination of the tegument; T4) Immersion in boiling water; T5) Alternating hot and cold water; T6) Immersion in concentrated H 2 SO 4 for 15 min; T7) Immersion in concentrated H 2 SO 4 for 30 min; T8) Stratification in wet and cold paper; T9) Stratification in moist sand and cold. After each treatment the seeds were placed in Petri dishes containing distilled water at 30°C temperature. Germination percentages (GP) were determined during 30 days. We used 20 seeds by treatment, with three replications each one. ABA and JA were extracted and purified from both Jatropha species tegument. These hormones were identified and quantified from tissue using reverse-phase high-performance liquid chromatography (HPLC)-mass spectrometry (MS). The total removal of tegument showed a 50% increase in germination percentage, with the other treatments achieved between 0-10%. JAs were the most abundant compound detected in tegument. ABA level was higher in J. curcas (628%) than in J. macrocarpa , for this reason we assume that the tegument ABA level is not directly linked to germination and/or dormancy of these Jatropha species. In contrast, level of JAs was higher in J. macrocarpa (101%) than in J. curcas. In effects JA could have a roll in inhibition of germination of J. macrocarpa seeds.


INTRODUCTION
The genus Jatropha (Euphorbiaceae) includes 172 species native to Central America and is also widely distributed in Africa, Asia and South America. In Argentina, it is reported that 11 native species of Jatropha include J. curcas L. and J. macrocarpa Griseb. These plants are perennial deciduous shrub, with the greatest importance mainly from its biofuel potential (Tang et.al, 2011).
J. curcas and J. macrocarpa growing in semi-arid and arid soils, and their non-edible seeds have high oil content (Achten et al., 2008).
While that J. curcas has aroused much interest worldwide as a new oleaginous crop for biodiesel, it is not a suitable crop for arid zones and plants are sensitive to frost (Andrade et al., 2008) and need annual rainfall is greater than 700 mm for good fruit production (Achten et al., 2008). In arid areas with winter frost, the species J. macrocarpa could be an interesting alternative because its natural distribution area presents in such climatic conditions .
To achieve a good production of a crop, it is essential to know the ability of the species to successfully complete two critical stages in the life cycle such as germination and seedling establishment. On the other hand, the plants have evolved seed dormancy, a temporal suppression of germination under the conditions favorable to germination; which ensures that seeds germinate at the appropriate time. Dormancy is a complex trait because it is influenced by both environmental and endogenous factors. Moreover, the final level of dormancy is determined by the contributions of the different tissues that comprise a seed; between them the seed coat (Lee et al., 2010) (Smykal et al., 2014. Induction of seed dormancy during the maturation stage and its release at a dry state after a certain period of time, which is called "after-ripening", is widespread phenomena observed in diverse species of seed plants (Beuley et al., 2013). In fact, in various species the mechanisms related to dormancy imposed by the seed head are related to restrictions the permeability of water and/or oxygen, with the existence of a mechanical resistance to the protrusion of the radicle, with the presence of inhibitors and/or the inability to leach inhibitors from the embryo Debeaujon et al., 2000) (Finch-Savage andLeubner-Metzger, 2006). Studies previous showed high variation in J. curcas seeds germination (Ginwall et al., 2005) (Ahamad et al., 2013). This variation is influenced by genotype, age, storage conditions of the seed and environmental conditions of the crop (Islam et al., 2009) (Pompelli et al., 2010) (Windauer et al., 2012) (Duong et al., 2013) (Moncaleano-Scandon et al., 2013. The germination rate decreases with age and with the storage of seed, this strongly affects the content of reserve substances in seeds and low germination rate (Moncaleano-Escandon et al., 2013). Also, it has been reported that the mechanical rupture of the tegument as a pre-planting treatment significantly increased seed germination and slightly stimulated the growth of J. curcas seedlings (Marcello et al., 2015) J. curcas and J. macrocarpa present a hard seminal covering that encloses the endosperm and the embryo. Several authors consider that this tegument is one of the factors that induce dormancy in J.
curcas (Windauer et al., 2012) (Mohan et al., 2011); however, in the J. macrocarpa the effects of the tegument in the low germinative power, is not yet studied. Several plant hormones play a role in dormancy and germination control (Linkies and Leubner-Metzger, 2012) (Arc et al., 2013).
Abscisic acid (ABA) is one of such hormone; that plays a prominent role in dormancy and germination control in coordinated interaction with various others (Nonogaky et al., 2014) (Nambara et al., 2010) (Shu et al., 2016). Recently, evidences have been provided for an interaction between ABA and jasmonates (JAs) in the regulation of these processes (Dave et al., 2016) (Xu et al., 2016). In particular, in ABA-JA interaction, Dave et al. (2016) confirmed that Arabidopsis thaliana seed dormancy is correlated with the accumulation level of oxo-phytodienoic-acid (OPDA), which acts synergistically with ABA, ABI5 transcription factor, DELLA RGL2 protein and MFT dormancy promoting factor in regulation of this process. On the contrary, Xu et al. (2016) reported that JAs and ABA have opposing roles in the regulation of dormancy release by stratification in wheat.
We hypothesize that the tegument is one of the factors that induce dormancy. The aim of the work was to study the role of tegument and abscisic acid (ABA) y jasmonic acid (JA) in dormancy and germination in these species.

MATERIAL AND METHODS
Seeds of J. macrocarpa were collected in a wild population located 30 km south of La Rioja city, Argentina (29.3˚S; 66.8˚W, 438 m above sea level) while the seeds of J. curcas were obtained from experimental plots located in Siete Palmas, Formosa, Argentina (58˚17'59.67"W -25˚13'21.04"S).

Seed morphology
Ten J. macrocarpa and J. curcas seeds were imbibed in distilled water for 24 h to facilitate removal of tegument to observe the embryo and nutritive tissues and ten endosperm of each species.

Seed treatments to break dormancy in J. macrocarpa
The seeds of J. macrocarpa were subjected to different scarification and stratification treatments: T1) Control; T2) Scarification with sandpaper; T3) Total elimination of the tegument; T4) Immersion in boiling water for 1 min and then immersed in cold water for 24 h; T5) Alternating hot and cold water for 5 min each one T6) Immersion in concentrated H2SO4 for 15 min; T7) Brazilian Journal of Animal and Environmental Research, Curitiba, v.5, n.4, p. 4101-4114, out./dez., 2022 Immersion in concentrated H2SO4 for 30 min; T8) Stratification in wet and cold paper (4˚C) for 90 days; T9) Stratification in moist sand and cold (4˚C) for 90 days. After each treatment the seeds were immediately placed on filter paper in Petri dishes containing 3 ml of distilled water at 30˚C temperature. The test was conducted under dark condition. Germination percentages (GP) were determined during 30 days. We used 20 seeds by treatment, with three replications each one.
The seeds of J. curcas don't were subjected to different treatments scarification and stratification because they haven't dormancy.

Extraction and purification of endogenous hormones
ABA and JA were extracted from both Jatropha species tegument using a modification of the protocol of Durgbanshi et al. (2005). 200 mg of tegument were homogenized in a mortar with liquid nitrogen and 5 ml ultra-pure water. D6-ABA (NRC-Plant Biotechnology Institute, Saskatoon, Canada) and D6-JA (Leibniz-Institute of Plant Biochemistry, in Halle, Germany) were used as internal standards. Extracts were transferred to 50-ml tubes, centrifuged at 1500× g for 15 min, pH of the supernatant was adjusted to 2.8 with 15% acetic acid, and supernatant was partitioned twice against an equal volume of diethyl ether. The aqueous phase was discarded, and the organic fraction was evaporated under vacuum. Dried extracts were dissolved in 1 ml methanol. Samples were filtered through a syringe filter tip on a vacuum manifold at flow rate < 1 ml min−1, and the eluate was evaporated at 35˚C under vacuum in a SpeedVac SC110 (Savant Instruments Inc. NY, USA).
The assay employed four biological replicates.

Hormone identification and quantification with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS-MS)
ABA and JAs were separated from tissue using reverse-phase high-performance liquid chromatography (HPLC). An Alliance 2695 separation module (Waters, Milford, MA, USA) equipped with a RestekC18 column (100 m × 2.1 mm, 3 μm) was used to maintain performance of the analytical column. Fractions were separated using a gradient of increasing methanol concentration, constant glacial acetic acid concentration (0.2% in water) and initial flow rate 0.2 ml•min−1. The gradient was increased linearly from 40% methanol/60% water-acetic acid at 25 min, to 80% methanol/20% water-acetic acid. After 1 min, the initial conditions were restored, and The collision energies used were 20 eV for JAs and ABA, and the cone voltage was 35 V. The spec spectrometry software used was Mass Lynx version 4.1 (Micromass).

Statistical analysis
Analysis of variance (ANOVA) was applied and data were subjected to Mul-tiple Range the Duncan. Test using the software INFOSTAT-UNC.

External and internal seed morphology
Seeds of J. curcas are oblong in shape with a convex dorsal area along which the raphe is visualized and in the hilar region a small conical caruncle of ivory color is observed (Figure 1(a), a). The average measurements of these seeds are: length 1.8 ± 0.03 cm, width 1.0 ± 0.01 cm and thickness 0.8 ± 0.02 cm. The tegument is very dark brown, smooth with porous texture and with small cracks that are more evident in the ventral zone. In this area, in the center of the caruncle the micropyle is observed (Figure 1(a), b). The tegument of J. macrocarpa is also smooth but light brown, mottled with dark brown. The seeds are subspherical, on average they are 1.5 ± 0.05 cm long, 1.3 ± 0.04 cm wide and 0.9 ± 0.02 cm thick. The dorsal area is slightly convex and is traversed by an evidentraphe (Figure 1(a), c) in these seeds the ventral zone is the most convex and to the hilar zone end is inserted. With a prominent caruncle that forms a ridge with irregular edges, the micropyle can be seen at the base of the crest (Figure 1(a), d). The embryo of J.
curcas has a cylindrical radicle of approximately 6 mm in length and two whitish ovoid, foliate, cotyledons that are inserted into the embryonic axis knot at its base. The blade of these leaves are thin and show a trinervia venation as it presents three main nerves that are born from the base of the blade foliar, two of them open laterally. The ribs are very marked on both the abaxial and adaxial sides (Figure 1(b)). In J. macrocarpa the embryo is smaller than in J. curcas, the radicle reaches 3 mm in length and its apex is markedly conical, its cotyledons are broad-bodied with apex rounded.
The blade is fleshy and also trinervia although the ribs are less evident than in J. curcas ( Figure   1(b)). In both species the cotyledons are faced for their adaxial face protecting the sheepish, and are externally surrounded by the nutrient tissue that in these seeds is the endosperm. This nutrient tissue is strongly attached to the embryo and has on its outer surface the impression of the radicle and the veins running along the abaxial surface of each cotyledon blade (Figure 1(c)). On the outside the endosperm is protected by a whitish membrane that is in contact with the tegument. The protective membrane of the seed of J. curcas is thicker and is furrowed by a set of important veins that leave their imprint on the inner side of the endosperm to which it covers firmly (Figure 1(d)). This membrane in J.macrocarpa is very tenuous and although this innervate does not form remarkable grooves in the surface of the same (Figure 1(d)).

Breakdormancy
J. macrocarpa present dormancy since it does not germinate by traditional methods. The effect of the different treatments to break seed dormancy of J. macrocarpa is showed in Figure   2. The total removal of tegument showed a 50% increase in germination percentage, with the other treatments achieved between 0% -10%. The seeds of J. curcas germinate without treatments for that reason the treatment was done only in the seeds of J. macrocarpa.

ABA and JAs level in Jatropha tegument
ABA and JAs were detected in tegument of J. macrocarpa and J. curcas seeds. JAs were the most abundant compound. Level of JAs was higher in J. macrocarpa (101%) than in J. curcas.

DISCUSSION
External morphology of the seeds of J. curcas and J. macrocarpa allow different them easily since they vary in size, shape and coloration of the tegument and the caruncle. Internally also the embryos show evident differences in special, in form, size and thickness of the foliar cotyledons and in length and shape of the radicle. Nevertheless, both species are endosperm and this tissue surrounds firmly to the embryo. The internal structure of these seeds is unusual in dicotyledons, but is common in Euphorbiaceae. A similar organization has been described for the seeds of other species of this family as Ricinus communis L. (Sing, 1954), Croton floribundus Spreng y Croton urucurana Baill (Paoli et al., 1995), Jatropha elliptica Mull. Arg. (Añes et al., 2005) and Cnidosculus juercifolius Paxe K. Hoffm (Silva et al., 2007).
Morphological characteristics found in the seeds of J. curcas were in many aspects coincident with those described previously by Loureiro et al., (2013)  for these authors is cordiform, with the narrow apex and a broad base excavated and rounded while for us they are ovoid.
The protective seed membrane that is located between the tegument and that tightly binds to the endosperm in J. curcas, it carry microorganisms that will hamper the seed germination (Mohan et al., 2011). Nevertheless, the tegument is a major barrier to radicle protrusion for many seeds (Zan et al., 2008), whose physical properties determine its effect on seed germination (Debeaujon et al., 2000). Our results showed that germination percentage of J. macrocarpa seeds with intact tegument was very low (4%). However, when the tegument was removed completely increased GP (from 4% to approximately 50%). These results indicate the presence of physical dormancy in J. macrocarpa seeds. In fact, it was reported that mechanical or chemical scarification can break physical seed dormancy (Finch-Savage and Leubner-Metzger, 2006). Similarly, Zhang et al. (2008) demonstrated that the tegument of canola (Brassica napus) restricted seed germination at low temperature and this inhibitory effect was more apparent in the yellow seed line compared to the black seed line. It is possible that differences in color of tegument of J. curcas and J. macrocarpa are also related to the differential level of dormancy observed between these seeds. In many species, the presence of tegument pigmentation color is associated with a different degree of permeability and dormancy (Mac Gregor et al., 2015).
The tegument eliminated of J. macrocarpa increase the germination of seeds, from 4% to 50%, so the tegument is directly relationally with de dormancy, although other tissues could be involved in the imposition of dormancy. This is a clear example that dormancy in some seeds resides in their teguments with probable intervention of the hormones, JA in this particular case. In this sense, the seed dormancy can be imposed by the embryo, the envelopes (tegument, endosperm, etc.), or a combination of both factors to an extent that depends on the plant species (Bewley, 1997).
Recent physiological and molecular studies have shown that physiological dormancy includes an embryo and coat component, and their sum and interaction determine the degree of whole-seed physiological dormancy (Finch-Savage and Leubner-Metzger, 2006). In fact, the dormancy attributed to different tissues of the seed has been reported in different species (Brunick, 2007) (Gu et al., 2015).
Hormones found in the dry seed are generally provided from the mother plant during seed maturation; in some cases, hormones leak from the embryo during late embryogenesis (Finkelstein et al., 2002). On the other hand, the mechanisms that lead to the definition of the structures composing the seed are highly coordinated and extremely complex and they involve a tight hormonal control and a continuous interchange of signals from and to the maternal tissues (Loscacio et al., 2014). There is considerable evidence that ABA is an important positive regulator of both the induction of dormancy and their maintenance (Nambara et al., 2010). We found that the tegument of J. macrocarpa dry seed have a significantly lower ABA content than J. curcas, for this reason we assume that the tegument ABA level is not directly linked to germination and/or dormancy of these Jatropha species. Indeed, in Arabidopsis thaliana the final ABA levels present in mature dry seeds are unrelated to the depth of dormancy (Lee et al., 2010) (Ali-Rachedi et al., 2004) that suggest that ABA abundance or signaling, or both, play an indirect role in promoting seed dormancy during seed development (Chahtane et al., 2017). In contrast, different studies have shown that the seed structures surrounding the embryo contain compounds possessing germination inhibitory activities including ABA (Bewley et al., 2013). For example, Jin et al. (1995) showed that high concentrations of ABA in pericarp and seed coat of rose achene could be inhibiting germination. Respect to JAs, there is no functional evidence supporting a role for a correlation between endogenous content in dry seed and level of seed dormancy. For example, Preston et al. 2009 showed that in dry seed of Arabidopsis thaliana, the JAs content in non dormant seeds was ten-fold higher than in dormant

CONCLUSION
This study has demonstrated the characteristics of dormancy of both species proposed for the production of biodiesel. It was shown that J. macrocarpa have physical dormancy. These studies are fundamental to face studies in relation to the crop establishment.