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Germination of Crotalaria and Lupinus (Fabaceae) seeds submitted to different pre-germination treatments and their effect on enzymatic activity during early germination

Germinação de sementes de Crotalaria e Lupinus (Fabaceae) submetidas a diferentes tratamentos pré-germinativos e o efeito da atividade enzimática na fase inicial da germinação

Abstract

Most of the wild and native legume seeds has a hard and impermeable testa, which causes physical dormancy and prevents them from germinating even when environmental conditions are favorable. The study evaluated the effect of scarification treatments on germination and enzymatic activity of Crotalaria longirostrata (Cl) and Lupinus exaltatus (Le) seeds. After scarification treatments, germination percentage (GP) and rate (GR) were assessed during 30 days after seeding (DAS); and water absorption (WA) and specific enzymatic activity (SEA) during early germination (0, 6, 18, 36, 72, 120 h) in a growing chamber at 25 °C and photoperiod of 12 h. Scarification with 98% H2SO4 15 min increased GP and GR in both species. At 30 DAS, GP and GR of Le seeds were 34% and 0.97 seeds day-1, respectively. In Cl seeds, GP was 64% and GR 0.90 seeds day-1. Scarification with H2O at 80 °C 1 min also promoted germination in Cl (52%). At 120 h after seeding, Le and Cl seeds showed already a high GP with acid scarification (31% and 48%, respectively). In seeds of both species, scarification treatments affected WA and SEA during early germination. During this period, scarification treatments that increased GP also showed a higher α-D-galactosidase activity. The maximum enzyme activity was observed 72 h after hot water scarification in Cl (82.6 U/mg total protein), followed by acid scarification (54.5 U/mg total protein). In Le, the activity peak was 36 h after acid scarification (9.5 U/mg total protein). No relationship was observed between β-glucosidase activity and GP in both species. In conclusion, during early germination of both species, the increase in GP is accompanied by a rise in α-D-galactosidase activity between 36 and 72 h after seeding; and in Cl seeds, an alternative scarification treatment to increase GP may be the use of hot water.

Keywords:
scarification; native legumes; enzymes

Resumo

A maioria das sementes de leguminosas nativas e selvagens têm um tegumento rígido e impermeável, ​​o que causa dormência física e impede a germinação, mesmo se as condições ambientais forem favoráveis. O estudo avaliou o efeito de tratamentos de escarificação sobre a germinação e a atividade enzimática de sementes de Crotalaria longirostrata (Cl) e Lupinus exaltatus (Le). Após os tratamentos de escarificação, a percentagem (PG) e a velocidade de germinação (VG) foram avaliadas durante 30 dias após a semeadura (DAS); absorção de água (AA) e atividade enzimática específica (AEE) na fase inicial da germinação (0, 6, 18, 36, 72 e 120 h) em uma câmara de crescimento a 25 °C e fotoperíodo de 12 h. A escarificação com 98% de H2SO4, durante 15 min aumentou PG e VG nas duas espécies. Aos 30 DDS, PG e VG de sementes de Le foram de 34% e 0,97 sementes dia -1, respectivamente. Em sementes de Cl, PG foi de 64% e VG 0,90 sementes dias-1. A escarificação com H2O a 80 °C 1 min também promoveu a germinação em Cl (52%). A 120 h após a semeadura, as sementes de Cl e Le já tinha atingido uma alta PG com escarificação ácida (31% e 48%, respectivamente). Nas sementes das duas espécies, os tratamentos de escarificação afetaram a AEE e a AA nafase inicial da germinação. Durante este período, os tratamentos de escarificação que aumentaram PG, também mostraram a atividade mais elevada de α-D-galactosidase. A atividade enzimática máxima foi observada 72 h após o tratamento com água quente em Cl (82,6 U/mg de proteína total), seguido por escarificação ácida (54,5 U/mg de proteína total). Em Le, o pico de atividade foi de 36 h após a aplicação do tratamento ácido (9,5 U/mg de proteína total). Em contraste, não foi observado nenhuma relação entre a actividade β-glicosidase e PG. Em conclusão, durante a germinação precoce das duas espécies, o aumento da GP é acompanhado por um aumento da atividade da α-D-galactosidase entre 36 e 72 h após a semeadura; e em sementes de Cl, um tratamento de escarificação alternativo para aumentar GP pode ser o uso de água quente.

Palavras-chave:
escarificação; leguminosas nativas; enzimas

1. Introduction

Legumes are essential components of ecosystems and crop systems because of the biological capacity to fix atmospheric nitrogen. They have a vital role in food security and in resilience from effects of climate change ( Liew et al., 2014 LIEW, L.C., SINGH, M.B. and BHALLA, P.L., 2014. Unique and conserved features of floral evocation in legumes. Journal of Integrative Plant Biology, vol. 56, no. 8, pp. 714-728. http://dx.doi.org/10.1111/jipb.12187. PMid:24930396.
http://dx.doi.org/10.1111/jipb.12187 ...
). In Mexico, they constitute the second largest family of Phanerogamae ( Sousa and Delgado, 1998 SOUSA, S.M. and DELGADO, S.A., 1998. Leguminosas mexicanas: fitogeografía, endemismo y orígenes. In: T.P. RAMAMOORTHY, R. BYE, A. LOT and J. FA, eds. Diversidad biológica de México: orígenes y distribución . México: Instituto de Biología, UNAM, pp. 449-500. ), and a high percentage are semi-domesticated or are still wild ( Fraile et al., 2007 FRAILE, M.E., GARCÍA-SUÁREZ, M.D., MARTÍNEZ-BERNAL, A. and SLOMIANSKI, R., 2007. Nutritivas y apetecibles: conozca de leguminosas comestibles. Parte I: hojas, vainas y semillas. Contactos, vol. 66, pp. 27-35. ).

A limitation for the agronomic use or ecological restoration of most of the legume seeds is that they do not germinate even when environmental conditions are favorable because of their hard, impermeable testa, which causes physical dormancy ( Jeller et al., 2003 JELLER, H., PEREZ, S.C.J.G.A. and RAIZER, J., 2003. Water uptake, priming, drying and storage effects in Cassia excelsa Schrad seeds. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 63, no. 1, pp. 61-68. http://dx.doi.org/10.1590/S1519-69842003000100008. PMid:12914415.
http://dx.doi.org/10.1590/S1519-6984200...
; Smýkal et al., 2014 SMÝKAL, P., VERNOUD, V., BLAIR, M., SOUKUP, A. and THOMPSON, R., 2014. The role of the testa during development and in establishment of dormancy of the legume seed. Frontiers in Plant Science, vol. 5, pp. 351. http://dx.doi.org/10.3389/fpls.2014.00351. PMid:25101104.
http://dx.doi.org/10.3389/fpls.2014.003...
). In breaking dormancy in this type of seeds, scarification treatments applied affect seed imbibition ( Bewley et al., 2013 BEWLEY, J.D., BRADFORD, K.J., HILHORST, H.W.M. and NONOGAKI, H., 2013. Seeds: physiology and dormancy. 3rd ed. New York: Springer. http://dx.doi.org/10.1007/978-1-4614-4693-4.
http://dx.doi.org/10.1007/978-1-4614-46...
). Application of acid or heat treatments can release physical dormancy in legumes seeds; and effectivity of these scarification treatments to promote germination range from 12 to 100% in Lupinus and Crotalaria species ( Elliott et al., 2011 ELLIOTT, C.A., FISCHER, D.G. and LE ROY, C.J., 2011. Germination of three native Lupinus species in response to temperature. Northwest Science , vol. 85, no. 2, pp. 403-410. http://dx.doi.org/10.3955/046.085.0223.
http://dx.doi.org/10.3955/046.085.0223 ...
; Alderete-Chávez et al., 2010a ALDERETE-CHÁVEZ, A., RODRÍGUEZ-TREJO, D.A., ESPINOSA-HERNÁNDEZ, V., OJEDA-TREJO, E. and DE LA CRUZ-LANDERO, N., 2010a. Effects of different scarification treatments on the germination of Lupinus leptophyllus seeds. International Journal of Botany, vol. 6, no. 1, pp. 64-68. http://dx.doi.org/10.3923/ijb.2010.64.68.
http://dx.doi.org/10.3923/ijb.2010.64.6...
, b ALDERETE-CHÁVEZ, A., AGUILAR-MARIN, L., DE LA CRUZ-LANDERO, N., GUERRA-SANTOS, J.J., BRITO, R., GUEVARA, E. and GELABERT, R., 2010b. Effects of scarification chemical treatments on the germination of Crotalaria retusa L. seeds. The Journal of Biological Sciences, vol. 10, no. 6, pp. 541-544. http://dx.doi.org/10.3923/jbs.2010.541.544.
http://dx.doi.org/10.3923/jbs.2010.541....
; Ayala-Herrada et al., 2010 AYALA-HERRADA, L., VILLAFAÑE-CRUZ, M., PINACHO-LOPEZ, B., ARROYO-LEDEZMA, J. and MAGAÑA-SEVILLA, H., 2010. Escarificación de semillas de Indigofera hirsuta (Linneaus), Canavalia maritima (Thouars) y Crotalaria longirostrata (Hook). Revista de la Facultad de Agronomía , vol. 27, pp. 433-446. ; Tiryaki and Topu, 2014 TIRYAKI, I. and TOPU, M., 2014. A novel method to overcome coat-imposed seed dormancy in Lupinus albus L. and Trifolium pratense L. Le Journal de Botanique, vol. 1, pp. 1-6. http://dx.doi.org/10.1155/2014/647469.
https://doi.org/10.1155/2014/647469...
).

Scarification treatments that break physical dormancy could have an effect on the composition, distribution of nutrients and enzymatic activity during dormancy release. In dormant seeds, the water content and metabolic rate is low, but a few enzymes are active, as α-galactosidase ( Guimarães et al., 2001 GUIMARÃES, V.M., REZENDE, S.T., MOREIRA, M.A., BARROS, E.G. and FELIX, C.R., 2001. Characterization of α-galactosidases from germinating soybean seed and their use for hydrolysis of oligosaccharides. Phytochemistry, vol. 58, no. 1, pp. 67-73. http://dx.doi.org/10.1016/S0031-9422(01)00165-0. PMid:11524115.
http://dx.doi.org/10.1016/S0031-9422(01...
; Fialho et al., 2008 FIALHO, L.S., GUIMARÃES, V.M., CALLEGARI, C.M., REIS, A.P., BARBOSA, D.S., BORGES, E.E.L., MOREIRA, M.A. and REZENDE, S.T., 2008. Characterization and biotechnological application of an acid a-galactosidase from Tachigali multijuga Benth. seeds. Phytochemistry, vol. 69, no. 14, pp. 2579-2585. http://dx.doi.org/10.1016/j.phytochem.2008.08.017. PMid:18834998.
http://dx.doi.org/10.1016/j.phytochem.2...
). The activity of α-galactosidase and other hydrolases increases during the polysaccharides hydrolysis in the early stages of seed germination ( Minic, 2008 MINIC, Z., 2008. Physiological roles of plant glycoside hydrolases. Planta , vol. 227, no. 4, pp. 723-740. http://dx.doi.org/10.1007/s00425-007-0668-y. PMid:18046575.
http://dx.doi.org/10.1007/s00425-007-06...
; Buckeridge, 2010 BUCKERIDGE, M.S., 2010. Seed cell wall storage polysaccharides: models to understand cell wall biosynthesis and degradation. Plant Physiology, vol. 154, no. 3, pp. 1017-1023. http://dx.doi.org/10.1104/pp.110.158642. PMid:20855518.
http://dx.doi.org/10.1104/pp.110.158642...
; Bewley et al., 2013 BEWLEY, J.D., BRADFORD, K.J., HILHORST, H.W.M. and NONOGAKI, H., 2013. Seeds: physiology and dormancy. 3rd ed. New York: Springer. http://dx.doi.org/10.1007/978-1-4614-4693-4.
http://dx.doi.org/10.1007/978-1-4614-46...
). The application of scarification treatments, weaken the cell walls, allowing to water enter; the seed hydrated increases metabolic activity, particularly enzyme in the endosperm or cotyledons for synthesis of new materials and embryo growth ( Bewley et al., 2013 BEWLEY, J.D., BRADFORD, K.J., HILHORST, H.W.M. and NONOGAKI, H., 2013. Seeds: physiology and dormancy. 3rd ed. New York: Springer. http://dx.doi.org/10.1007/978-1-4614-4693-4.
http://dx.doi.org/10.1007/978-1-4614-46...
; Kamithi et al., 2016 KAMITHI, K.D., WACHIRA, F. and KIBE, A.M., 2016 [viewed 26 September 2017]. Effects of different priming methods and priming durations on enzyme activities in germinating chickpea ( Cicer arietinum L.). American Journal of Natural and Applied Sciences [online], vol. 1, pp. A1-A9. Available from: http://www.ASRAresearch.org/ajnas-vol-1-no-1-2016
http://www.ASRAresearch.org/ajnas-vol-1...
). In Euphorbia heterophylla L. seeds, high levels of α-D-galactosidase are detected during the period of imbibition, related to reduction of endosperm resistance to cotyledon expansion ( Suda et al., 2003 SUDA, N.K.C., BUCKERIDGE, M.S. and GIORGINI, J.F., 2003. Cell wall hydrolases in the seeds of Euphorbia heterophylla L. during germination and early seedling development. Brazilian Journal of Plant Physiology, vol. 15, no. 3, pp. 135-143. http://dx.doi.org/10.1590/S1677-04202003000300002.
http://dx.doi.org/10.1590/S1677-0420200...
). In L. angustifolius this enzyme catalyzes hydrolysis of galactans in the cell walls of cotyledons during early stages of germination ( Buckeridge et al., 2005 BUCKERIDGE, M.S., HUTCHEON, I.S. and REID, J.S.G., 2005. The role of exo-(1,4)-β-galactanase in the mobilization of polysaccharides from the cotyledon cell walls of Lupinus angustifolius following germination. Annals of Botany, vol. 96, no. 3, pp. 435-444. http://dx.doi.org/10.1093/aob/mci192. PMid:15994843.
http://dx.doi.org/10.1093/aob/mci192 ...
), and in Arabidopsis seeds ß-glucosidase, seems to be involved in the embryo cell wall loosening needed for cell elongation and radicle extension ( Gallardo et al., 2002 GALLARDO, K., JOB, C., GROOT, S.P.C., PUYPE, M., DEMOL, H., VANDEKERCKHOVE, J. and JOB, D., 2002. Proteomics of Arabidopsis seed germination: a comparative study of wild-type and gibberellin-deficient seeds. Plant Physiology, vol. 129, no. 2, pp. 823-837. http://dx.doi.org/10.1104/pp.002816. PMid:12068122.
http://dx.doi.org/10.1104/pp.002816 ...
).

Species from the Lupinus and Crotalaria genus are abundant in ecosystem of Mexico ( Acosta-Percástegui and Rodríguez-Trejo, 2005 ACOSTA-PERCÁSTEGUI, J. and RODRÍGUEZ-TREJO, D.A., 2005 [viewed 26 September 2017]. Factors affecting germination and pregerminative treatments of Lupinus montanus seeds. Interciencia [online], vol. 30, pp. 576-579. Available from: http://www.redalyc.org/articulo.oa?id=33910811
http://www.redalyc.org/articulo.oa?id=3...
; Ayala-Herrada et al., 2010 AYALA-HERRADA, L., VILLAFAÑE-CRUZ, M., PINACHO-LOPEZ, B., ARROYO-LEDEZMA, J. and MAGAÑA-SEVILLA, H., 2010. Escarificación de semillas de Indigofera hirsuta (Linneaus), Canavalia maritima (Thouars) y Crotalaria longirostrata (Hook). Revista de la Facultad de Agronomía , vol. 27, pp. 433-446. ; Lagunes-Espinoza et al., 2012 LAGUNES-ESPINOZA, L.C., LÓPEZ-UPTON, J., GARCÍA-LÓPEZ, E., JASSO-MATA, J., DELGADO-ALVARADO, A. and GARCÍA DE LOS SANTOS, G., 2012. Diversidad morfológica y concentración de proteína de Lupinus spp. en la región centro-oriental del estado de Puebla, México. Acta Botánica Mexicana , vol. 99, no. 99, pp. 73-90. http://dx.doi.org/10.21829/abm99.2012.20.
http://dx.doi.org/10.21829/abm99.2012.2...
; Soto-Correa et al., 2015 SOTO-CORREA, J.C., SÁENZ-ROMERO, C., HORACIO, H. and LINDIG-CISNEROS, R., 2015. Estrés por sequía en Lupinus elegans procedentes de diferentes altitudes. Madera y Bosques, vol. 21, no. 1, pp. 35-43. http://dx.doi.org/10.21829/myb.2015.211431.
http://dx.doi.org/10.21829/myb.2015.211...
). Seed dormancy of these legumes can be broken by physical ( Pablo-Pérez et al., 2013 PABLO-PÉREZ, M., LAGUNES-ESPINOZA, L.C., LÓPEZ-UPTON, J., RAMOS-JUÁREZ, J. and ARANDA-IBÁÑEZ, E.M., 2013. Morfometría, germinación y composición mineral de semillas de Lupinus silvestres. Bioagro-, vol. 25, pp. 101-108. ) or chemical treatments ( Ayala-Herrada et al., 2010 AYALA-HERRADA, L., VILLAFAÑE-CRUZ, M., PINACHO-LOPEZ, B., ARROYO-LEDEZMA, J. and MAGAÑA-SEVILLA, H., 2010. Escarificación de semillas de Indigofera hirsuta (Linneaus), Canavalia maritima (Thouars) y Crotalaria longirostrata (Hook). Revista de la Facultad de Agronomía , vol. 27, pp. 433-446. ; Acosta-Percástegui and Rodríguez-Trejo, 2005 ACOSTA-PERCÁSTEGUI, J. and RODRÍGUEZ-TREJO, D.A., 2005 [viewed 26 September 2017]. Factors affecting germination and pregerminative treatments of Lupinus montanus seeds. Interciencia [online], vol. 30, pp. 576-579. Available from: http://www.redalyc.org/articulo.oa?id=33910811
http://www.redalyc.org/articulo.oa?id=3...
). Water absorption by seeds after scarification treatments enhances mobilization of nutrients and enzyme activity, required for rapid seed germination, so that scarification treatments could have a beneficial effect on enzyme activities involved in early germination ( Kamithi et al., 2016 KAMITHI, K.D., WACHIRA, F. and KIBE, A.M., 2016 [viewed 26 September 2017]. Effects of different priming methods and priming durations on enzyme activities in germinating chickpea ( Cicer arietinum L.). American Journal of Natural and Applied Sciences [online], vol. 1, pp. A1-A9. Available from: http://www.ASRAresearch.org/ajnas-vol-1-no-1-2016
http://www.ASRAresearch.org/ajnas-vol-1...
). In Lupinus and Crotalaria, few studies have focused on the effect of scarification treatments in the increase of enzymes activity during early germination that can be relationship with germination. In the present study, the effect of scarification treatments on percentage and rate of germination during time and specific enzyme activity of β-glucosidase and α-D-galactosidase in early germination were studied in C. longirostrata and L. exaltatus seeds.

2. Material and Methods

Dry fruits of L. exaltatus L. were collected in August 2014 (summer season), and those of Crotalaria longirostrata Hook. & Arn. fruits in September 2013 (autumn season), from plants grown in agroecosystems of the states of Puebla and Tabasco, Mexico, respectively. The seeds were separated from the dry fruits and conserved at 4 °C for later analysis.

Before application of scarification treatments, seeds were disinfected 2 min with a 3% solution of sodium hypochlorite, and washed three times with sterile distilled water. After seed disinfection, seeds of both species were tested for viability. 20 seeds in triplicate per species were disinfected and mechanically scarified to separate the testa from the cotyledons. The cotyledons were submerged in 5 mL of a 1% tetrazolium chloride solution and left in darkness for 24 h. The embryos dyed red (viable seeds) were counted. Seed viability was 93±5.7% and 90±5.0% for C. longirostrata and L. exaltatus, respectively.

After this step, other seeds were used for the scarification treatments. The scarification treatments applied to the seeds of L. exaltatus were: I) Immersion in H 2SO4 98% for 15 min followed by three washes with sterile distilled water; II) Immersion in wet sand for 8 h at 35 °C, followed by 16 h at room temperature (23-25 °C); III) Drying seeds at 80 °C for 7 min, then allowed to cool to room temperature; IV) Drying seeds in dry sand at 150 °C for 1 min; V) Without scarification (control). In C. longirostrata seeds the treatments applied were: I) Immersion in H2 SO4 98% for 15 min followed by three washes with sterile distilled water; II) Soaking in distilled water at 80 °C for 1 min; III) Without scarification (control).

After treatments, groups of 50 seeds in triplicate were sown in Petri dishes disinfected between sterile paper and placed in a bioclimatic chamber (Thermo Scientific) at 25 °C and 12 h light photoperiod ( Acosta-Percástegui and Rodriguez-Trejo, 2005 ACOSTA-PERCÁSTEGUI, J. and RODRÍGUEZ-TREJO, D.A., 2005 [viewed 26 September 2017]. Factors affecting germination and pregerminative treatments of Lupinus montanus seeds. Interciencia [online], vol. 30, pp. 576-579. Available from: http://www.redalyc.org/articulo.oa?id=33910811
http://www.redalyc.org/articulo.oa?id=3...
; Gutierrez-Nava et al., 2010 GUTIÉRREZ NAVA, P., DE LEÓN GONZÁLEZ, F., ETCHEVERS BARRA, J. and CASAS FERNÁNDEZ, A., 2010. Effect of scarification, self-inhibition, and sowing depth on seed germination of Lupinus campestris. Chilean Journal of Agricultural Research, vol. 70, no. 3, pp. 365-371. http://dx.doi.org/10.4067/S0718-58392010000300003.
http://dx.doi.org/10.4067/S0718-5839201...
). All lots of seeds were watered with sterile distilled water during the evaluation period. In a separate group of 50 seeds in triplicate, an imbibition test was performed after scarification treatments during 0, 6, 18, 36, 72, 120 and 148 h.

Germination percentage (GP) was determined with the formula of Maguire (1962) MAGUIRE, J.D., 1962. Speed of germination: aid in selection and evaluation for seedling emergence and vigour. Crop Science, vol. 2, no. 2, pp. 176-177. http://dx.doi.org/10.2135/cropsci1962.0011183X000200020033x.
http://dx.doi.org/10.2135/cropsci1962.0...
: GP%=NGSTNSS×100, where NGS = number of germinated seeds and TNSS = total number of sown seeds. For the germination test, germinated seeds were counted daily during 30 days or 0, 6, 18, 36, 72, and 120 h after seeding; a seed was considered germinated when the radicle was ≥ 2 mm long. Germination rate (GR) is defined as the measure of the number of seeds germinated relative to the time of germination, determined with the formula GR = ∑ (NGSi)/t, where NGSi = number of germinated seeds on day i; t = germination time from sowing to germination of the last seed.

To detect the peak of maximum enzyme activity during the first hours of seed imbibition, evaluation of enzymatic activity was at 0, 6, 18, 36, 72, and 120 h after seeding. To β-glucosidase enzyme activity, samples were powdered homogeneously in liquid N2; de-fatted to obtain acetone-dry powders (ADP) by addition of 95% hexane (1:10 w/v), with constant shaking (150 rpm) for two hours at 4 °C and centrifuged (10 000 rpm) for five min at 4 °C. The supernatant was decanted and the precipitate was left to stand until the solvent evaporated completely. Immediately, three extractions were done successively with 80% acetone (1:3 w/v); between each extraction, the sample was centrifuged (5,000 rpm) for 5 min at 4 °C. The supernatant was discarded. Finally, one extraction was done with 100% acetone (1:3 w/v) at 10,000 rpm for 5 min at 4 °C, discarding the supernatant; the residue was left at room temperature until the solvent evaporated completely. The resulting paste (ADP) was powdered and conserved at -67 °C. Thirty mg of polyvinylpolypyrrolidone (PVPP) and 900 μL of 1% Triton X100 solution (dissolved in 100 mM pH 7.0 of Tris ultrapure buffer) were added to 15 mg ADP and incubated at 37 °C in a water bath for 30 min with constant shaking at 150 rpm. The solution was then centrifuged (10,000 rpm) for 10 min at 4 °C. The supernatant was used to determine β-glucosidase enzyme activity. β-glucosidase activity was determined following Ross et al. (1993) ROSS, S.G., REDGWELL, R.J. and MACRAE, E.A., 1993. Kiwifruit β-galactosidase: Isolation and activity against specific fruit cell-wall polysaccharides. Planta , vol. 189, no. 4, pp. 499-506. http://dx.doi.org/10.1007/BF00198212.
http://dx.doi.org/10.1007/BF00198212 ...
and Gerardi et al. (2001) GERARDI, C., BLANDO, F., SANTINO, A. and ZACHEO, G., 2001. Purification and characterisation of a β-glucosidase abundantly expressed in ripe sweet cherry (Prunus avium L.) fruit. Plant Science, vol. 160, no. 5, pp. 795-805. http://dx.doi.org/10.1016/S0168-9452(00)00423-4. PMid:11297776.
http://dx.doi.org/10.1016/S0168-9452(00...
: 200 μL of the enzyme extract was mixed with 200 μL of 125 mM pH 4.0 sodium acetate buffer containing 200 mM 2-β-mercaptoethanol, 50 mL of 40 mM p-nitrophenyl β-D-glucopyranoside and 350 μL sterile deionized water. All was mixed in a vortex and placed in incubation at 30 °C for 60 min. The reaction was stopped by the addition of 200 μL 1 M Na 2CO3. In the blank samples, enzyme reaction was ended before incubation. Absorbance was determined at 405 nm in a Thermo Scientific model Multiskan Go spectrophotometer. The type curve was constructed using p-nitrophenol as standard at a concentration of 0 to 100 µM. One unit of β-glucosidase was defined as the quantity of enzyme capable of releasing one µM p-nitrophenol per minute.

To α-D-galactosidase enzyme activity, samples were powdered with liquid N2 , and homogenized with 20 mL 15 mM trihydrated sodium acetate buffer pH 5.5 solution. The homogenized solution was centrifuged (10,000 ppm) at 4 °C for five min ( Fialho et al., 2008 FIALHO, L.S., GUIMARÃES, V.M., CALLEGARI, C.M., REIS, A.P., BARBOSA, D.S., BORGES, E.E.L., MOREIRA, M.A. and REZENDE, S.T., 2008. Characterization and biotechnological application of an acid a-galactosidase from Tachigali multijuga Benth. seeds. Phytochemistry, vol. 69, no. 14, pp. 2579-2585. http://dx.doi.org/10.1016/j.phytochem.2008.08.017. PMid:18834998.
http://dx.doi.org/10.1016/j.phytochem.2...
). Finally, the supernatant was decanted and stored at -67 °C. Activity was determined following Fialho et al. (2008) FIALHO, L.S., GUIMARÃES, V.M., CALLEGARI, C.M., REIS, A.P., BARBOSA, D.S., BORGES, E.E.L., MOREIRA, M.A. and REZENDE, S.T., 2008. Characterization and biotechnological application of an acid a-galactosidase from Tachigali multijuga Benth. seeds. Phytochemistry, vol. 69, no. 14, pp. 2579-2585. http://dx.doi.org/10.1016/j.phytochem.2008.08.017. PMid:18834998.
http://dx.doi.org/10.1016/j.phytochem.2...
, using 100 μL enzyme extract, 600 μL 200 mM sodium acetate buffer pH 5, and 50 μL 20 mM p-nitrophenol α-D- galactopyranoside substrate. The reaction was run for 15 min at 40 °C and stopped with the addition of 250 μL 2 M sodium carbonate (Na2 CO3), centrifuged (10,000 rpm) for 5 min at room temperature. For the blanks, the enzyme reaction was stopped before incubation. The amount of p-nitrophenol released was determined at 410 nm. A standard curve of p-nitrophenol of 0 to 100 µM was constructed to interpolate the data. One unit of α-D-galactosidase was defined as the amount of enzyme capable of releasing one µM p-nitrophenol per minute.

The concentration of total protein in the enzymatic extracts was determined by the micro Markwell method ( Markwell et al., 1978 MARKWELL, M.A.K., HAAS, M., BIEBER, L.L. and TOLBERT, N.E., 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Analytical Biochemistry, vol. 87, no. 1, pp. 206-210. http://dx.doi.org/10.1016/0003-2697(78)90586-9. PMid:98070.
http://dx.doi.org/10.1016/0003-2697(78)...
). To 20 μL of extract, 280 μL sterile deionized water was added. While shaking, 1,000 μL of a solution composed of A [2% solution of anhydrous sodium carbonate (Na2CO3), 0.4% sodium hydroxide (NaOH), 0.16% sodium tartrate and potassium (C4H4KNaO6·4H2O), 1% sodium dodecyl sulfate (CH3 (CH2)11OSO3 Na)] was mixed with solution B [1:100 w/v of cuprous sulfate (CuSO4 ·5H2O)]. The mixture was incubated for 15 min at room temperature, and then while shaking 100 μL Folin & Ciocalteu’s reagent (1:1, reagent:water) were added and incubated for 5 min at 37 °C. The peptides released during the reaction were determined at 660 nm using bovine serum albumin (0 to 100 µg/µL).

Previous statistical analysis, data on germination percentage were transformed to the arcsine square root to have an approximated normal distribution. All data were subjected, by species, to analysis of variance under a completely randomized design in a factorial array with three replications. The first factor were the scarification treatments, and the second factor was time in days (30 days) or hours (0, 6, 18, 36, 72 and 120 h). The Tukey means test at 5% of probability was used to determine significant differences between treatments with SAS 9.3 software.

3. Results

The scarification pre-treatments affected germination percentage (GP) and rate (GR) of L. exaltatus and C. longirostrata seeds. Acid scarification pre-treatment increase germination percentage and rate in both species ( Figure 1 ). At 30 DAS of C. longirostrata seeds had germinated and only 34% in L. exaltatus. In both species, germination rate decreased with germination time, the highest was five DAS after acid pre-treatment (4.8 seeds day-1 in C. longirostrata and 3.8 in L. exaltatus). In C. longirostrata , the pre-treatment with moist heat also increased significantly GP and GR (52% and 3.4 seeds day-1, respectively), in relation to control five DAS.

Figure 1
Germination percentage of L. exaltatus (A) and C. longirostrata (B) seeds at 30 days after application of scarification treatments. Germination rate of L. exaltatus (C) and C. longirostrata (D) seeds during germination time after application of scarification treatments. Different letters by treatments are statistically different (Tukey, p≤0.05). Error bars represent ± s.e. (n=3).

Regarding the GP and GR during early germination, dry heat shock (80 °C and 150 °C) inhibited the germination process almost entirely in L. exaltatus ( Figure 2 2B). In contrast, pre-treatment of sulfuric acid 98% 15 min increased GP and GR in L. exaltatus seeds. At the end of 120 h, the seeds had already reached 31% germination. In contrast to L. exaltatus, the response to germination of C. longirostrata seeds to scarification pre-treatment was higher ( Figure 2 C). In this species, no significant differences (p≤0.0001) were observed between treatments with sulfuric acid 98% and water at 80 °C on the GR at 120 h. The higher GR was observed with sulfuric acid (0.37 seeds h-1) followed by treatment of immersion in water at 80 °C for 1 min (0.27 seeds h-1) at 36 h after seeding ( Figure 2 D). In GP, at 120 h, 48 and 55% of the seeds germinated after sulfuric acid 98% and water at 80 °C treatment, respectively.

Figure 2
Germination percentage and rate of L. exaltatus (A, B) and C. longirostrata (C, D) seed during early germination time (0, 6, 18, 36, 72 and 120 h after application of scarification treatments). Different letters by treatments are statistically different (Tukey, p≤0.05). Error bars represent ± s.e. (n=3).

Water absorption was faster in L. exaltatus seeds after application of sulfuric acid 98% for 15 min ( Figure 3 A), than in the other scarification pre-treatments during early germination. No seed weight gain was observed after application of dry heat at 150 °C treatment. In C. longirostrata , from 72 h after imbibition, water absorption increased after application of water at 80 °C for 1 min followed by sulfuric acid 98% for 15 min pre-treatment ( Figure 3 B). At this time, the increase in the growth of the embryo and radicle emergence affected seed weight due to as more than 50% of seeds began germination.

Figure 3
Accumulated seed weight (water absorption) of L. exaltatus (A) and C. longirostrata (B) after application of scarification treatments over time. *Start of germination. Error bars represent ± s.e. (n=3).

During early germination, in L exaltatus, heat shock pre-treatment (dry sand 150 °C) that completely inhibited GP, also inhibited enzyme activity ( Figure 4 4B). The α-D-galactosidase activity increased after acid pre-treatment to 36 h, and then declined ( Figure 4 A). The maximum peak activity of this enzyme was found at 36 h (55.6 U/mg total protein). Enzymatic activity of β-glucosidase was no significantly different (p ≥ 0.05) between the pre-treatments ( Figure 4 B). In C. longirostrata, the highest α-D-galactosidase activity was observed at 72 h with treatment of immersion in water at 80 °C 1 min ( Figure 4 C), followed by those of sulfuric acid 98% pre-treatment. At time 120 h, both pre-treatments showed close to or more than 50% germination ( Figure 2 C), just as the enzyme activity decreased significantly. Indicating that once most of seeds germinated, the activity of α-D-galactosidase decreased. The highest concentration of β-glucosidase activity was found in the seeds treated with sulfuric acid at time 0 (3.77 U/mg total protein) ( Figure 4 D).

Figure 4
Specific activity of α-D-galactosidase and β-glucosidase in L. exaltatus (A, B) and C. longirostrata (C, D) seeds during early germination time, after subjected to scarification treatments. Different letters by enzyme are statistically different (Tukey, p≤0.05). Error bars represent ± s.e. (n=3).

4. Discussion

Seeds of C. longirostrata and L. exaltatus need scarification to increase water absorption ( Ayala-Herrada et al., 2010 AYALA-HERRADA, L., VILLAFAÑE-CRUZ, M., PINACHO-LOPEZ, B., ARROYO-LEDEZMA, J. and MAGAÑA-SEVILLA, H., 2010. Escarificación de semillas de Indigofera hirsuta (Linneaus), Canavalia maritima (Thouars) y Crotalaria longirostrata (Hook). Revista de la Facultad de Agronomía , vol. 27, pp. 433-446. ; Pablo-Pérez et al., 2013 PABLO-PÉREZ, M., LAGUNES-ESPINOZA, L.C., LÓPEZ-UPTON, J., RAMOS-JUÁREZ, J. and ARANDA-IBÁÑEZ, E.M., 2013. Morfometría, germinación y composición mineral de semillas de Lupinus silvestres. Bioagro-, vol. 25, pp. 101-108. ) and initiate metabolic changes to carry out the germination process. Scarification with sulfuric acid 98% to soften the seed coat had a significant effect on the germination percentage and rate in the two species. In L. exaltatus GP was 31% at 120 h after seeding and 34% at 30 DAS. In studies with L. leptophyllus the application of this acid promoted 15% of germination at 120 h ( Alderete-Chávez et al., 2010a ALDERETE-CHÁVEZ, A., RODRÍGUEZ-TREJO, D.A., ESPINOSA-HERNÁNDEZ, V., OJEDA-TREJO, E. and DE LA CRUZ-LANDERO, N., 2010a. Effects of different scarification treatments on the germination of Lupinus leptophyllus seeds. International Journal of Botany, vol. 6, no. 1, pp. 64-68. http://dx.doi.org/10.3923/ijb.2010.64.68.
http://dx.doi.org/10.3923/ijb.2010.64.6...
). A higher germination percentage has been observed in other Lupinus species. In L. campestris seeds, sulfuric acid also broke dormancy, but with 90 min of exposure, peaking at 50% at 30 DAS (Gutierrez-Nava et al., 2010). Dry heat treatment did not promote germination in L. exaltatus from study area after 30 DAS. Nevertheless, L. exaltatus growing in areas prone to accidental fires in the state of Jalisco, Mexico, shows 93% of germination seven weeks after seeding, when exposed to heat shock at 150 °C 1 min ( Zuloaga-Aguilar et al., 2010 ZULOAGA-AGUILAR, S., BRIONES, O. and OROZCO-SEGOVIA, A., 2010. Effect of heat shock on germination of 23 plant species in pine–oak and montane cloud forests in western Mexico. International Journal of Wildland Fire, vol. 19, no. 6, pp. 759-773. http://dx.doi.org/10.1071/WF08092.
http://dx.doi.org/10.1071/WF08092 ...
). The results obtained suggest that a longer germination time could be necessary to evaluate these pre-treatments of dry heat in this species from Puebla, Mexico, although in the genus Lupinus has been observed a high variation in germination, in response to pre-germination treatments ( Elliott et al., 2011 ELLIOTT, C.A., FISCHER, D.G. and LE ROY, C.J., 2011. Germination of three native Lupinus species in response to temperature. Northwest Science , vol. 85, no. 2, pp. 403-410. http://dx.doi.org/10.3955/046.085.0223.
http://dx.doi.org/10.3955/046.085.0223 ...
).

Different studies in Crotalaria spp. have shown a positive effect of using acid sulfuric to release dormancy and promote germination. In C. longirostrata from Oaxaca, Mexico, application of sulfuric acid 98% for 60 min resulted in 99% of germination at seven days ( Ayala-Herrada et al., 2010 AYALA-HERRADA, L., VILLAFAÑE-CRUZ, M., PINACHO-LOPEZ, B., ARROYO-LEDEZMA, J. and MAGAÑA-SEVILLA, H., 2010. Escarificación de semillas de Indigofera hirsuta (Linneaus), Canavalia maritima (Thouars) y Crotalaria longirostrata (Hook). Revista de la Facultad de Agronomía , vol. 27, pp. 433-446. ), but in C. retusa with 20 min of acid exposure was only 20% at 5 DAS ( Alderete-Chávez et al., 2010b ALDERETE-CHÁVEZ, A., AGUILAR-MARIN, L., DE LA CRUZ-LANDERO, N., GUERRA-SANTOS, J.J., BRITO, R., GUEVARA, E. and GELABERT, R., 2010b. Effects of scarification chemical treatments on the germination of Crotalaria retusa L. seeds. The Journal of Biological Sciences, vol. 10, no. 6, pp. 541-544. http://dx.doi.org/10.3923/jbs.2010.541.544.
http://dx.doi.org/10.3923/jbs.2010.541....
). In Crotalaria seeds from Tabasco, Mexico it was observed 48% of germination at 5 DAS with sulfuric acid for 15 min. But using water 80 °C 1 min, the germination percentage was increased to 55% 5 DAS and >60% 30 DAS. Muñoz et al. (2009) MUÑOZ, C.B., SÁNCHEZ, J.A., MONTEJO, L.A., GONZÁLEZ, Y. and REINO, J., 2009. Valoración germinativa de 20 accesiones de leguminosas almacenadas en condiciones desfavorables. Pastos y Forrajes, vol. 32, pp. 1-15. observed a similar response in seeds of Crotalaria cv. right to acid treatment, despite seeds had more than 12 years of storage.

High specific activity of α-D-galactosidase was observed in seeds of both species as of five DAS. Kadlec et al. (2008) KADLEC, P., DOSTÁLOVÁ, J., BERNÁŠKOVÁ, J. and SKULINOVÁ, M., 2008. Degradation of α-galactosides during the germination of grain legume seeds. Czech Journal of Food Sciences, vol. 26, no. 2, pp. 99-108. http://dx.doi.org/10.17221/3/2008-CJFS.
http://dx.doi.org/10.17221/3/2008-CJFS ...
observed that in soybeans, peas and lupine seeds levels of α-galactosides decrease during the first two days of imbibition, and hydrolysis takes 4 to 5 days. In Tachigali multijuga ( Fialho et al., 2008 FIALHO, L.S., GUIMARÃES, V.M., CALLEGARI, C.M., REIS, A.P., BARBOSA, D.S., BORGES, E.E.L., MOREIRA, M.A. and REZENDE, S.T., 2008. Characterization and biotechnological application of an acid a-galactosidase from Tachigali multijuga Benth. seeds. Phytochemistry, vol. 69, no. 14, pp. 2579-2585. http://dx.doi.org/10.1016/j.phytochem.2008.08.017. PMid:18834998.
http://dx.doi.org/10.1016/j.phytochem.2...
) an increase in α-D-galactosidase activity was detected the first 108 h of imbibition. This enzyme catalyzes hydrolysis of galactans in the cell walls of L. angustifolius cotyledons during early stages of germination ( Buckeridge et al., 2005 BUCKERIDGE, M.S., HUTCHEON, I.S. and REID, J.S.G., 2005. The role of exo-(1,4)-β-galactanase in the mobilization of polysaccharides from the cotyledon cell walls of Lupinus angustifolius following germination. Annals of Botany, vol. 96, no. 3, pp. 435-444. http://dx.doi.org/10.1093/aob/mci192. PMid:15994843.
http://dx.doi.org/10.1093/aob/mci192 ...
). In this study, changes in the activity of enzymes tested occurred during the first hours of imbibition. Acid scarification treatment modified enzymatic activity of α-D-galactosidase during the first 120 h, and this increase was when the germination percentage was high. In C. longirostrata, the enzyme showed higher activity at 72 h after application of treatments. In L. exaltatus, at 36 h after sulfuric acid treatment. As previously indicated, this enzyme has the ability to hydrolyze both stachyose and raffinose sugars and galactomannans in the cell walls of cotyledons to support radicle emergence ( Bewley et al., 2013 BEWLEY, J.D., BRADFORD, K.J., HILHORST, H.W.M. and NONOGAKI, H., 2013. Seeds: physiology and dormancy. 3rd ed. New York: Springer. http://dx.doi.org/10.1007/978-1-4614-4693-4.
http://dx.doi.org/10.1007/978-1-4614-46...
; Buckeridge et al., 2005 BUCKERIDGE, M.S., HUTCHEON, I.S. and REID, J.S.G., 2005. The role of exo-(1,4)-β-galactanase in the mobilization of polysaccharides from the cotyledon cell walls of Lupinus angustifolius following germination. Annals of Botany, vol. 96, no. 3, pp. 435-444. http://dx.doi.org/10.1093/aob/mci192. PMid:15994843.
http://dx.doi.org/10.1093/aob/mci192 ...
). As increase α-galactosidase activity, raffinose and stachyose content decrease ( Fialho et al., 2008 FIALHO, L.S., GUIMARÃES, V.M., CALLEGARI, C.M., REIS, A.P., BARBOSA, D.S., BORGES, E.E.L., MOREIRA, M.A. and REZENDE, S.T., 2008. Characterization and biotechnological application of an acid a-galactosidase from Tachigali multijuga Benth. seeds. Phytochemistry, vol. 69, no. 14, pp. 2579-2585. http://dx.doi.org/10.1016/j.phytochem.2008.08.017. PMid:18834998.
http://dx.doi.org/10.1016/j.phytochem.2...
). The β-glucosidase activity did not show relation with germination percentage although several studies mentioned their involvement in germination process ( Singh et al., 2016 SINGH, G., VERMA, A.K. and KUMAR, V., 2016. Catalytic properties, functional attributes and industrial applications of β-glucosidases. 3 Biotech, vol. 6, no. 1, pp. 1-14. http://dx.doi.org/10.1007/s13205-015-0328-z. PMid:28330074.
http://dx.doi.org/10.1007/s13205-015-03...
; Minic, 2008 MINIC, Z., 2008. Physiological roles of plant glycoside hydrolases. Planta , vol. 227, no. 4, pp. 723-740. http://dx.doi.org/10.1007/s00425-007-0668-y. PMid:18046575.
http://dx.doi.org/10.1007/s00425-007-06...
).

In conclusion, sulfuric acid treatment increases GP and GR in both species. In seeds of C. longirostrata, scarification treatment using hot water may be an alternative scarification treatment as it also showed a high GP. During early germination of the seeds of both species, between 36 and 76 h after seeding the increase in GP is accompanied by a rise in α-D-galactosidase activity.

Acknowledgements

Authors are grateful to Consejo Nacional de Ciencia y Tecnología (CONACYT, project CB-2012-01 181428) and CP (project 449), for funding the study. The first author is grateful to CONACYT for the scholarship granted to pursue graduate studies.

  • (With 4 figures)

References

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Publication Dates

  • Publication in this collection
    18 Apr 2019
  • Date of issue
    Jan-Feb 2020

History

  • Received
    26 Sept 2017
  • Accepted
    16 May 2018
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