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Enzyme activity in the micropylar region of Melanoxylon brauna Schott seeds during germination under heat stress conditions

Atividade enzimática na região micropilar de sementes de Melanoxylon brauna Schott durante a germinação sob estresse térmico

Abstract:

Recent studies indicate that global temperatures will rise substantially in the 21st century, leading to the extinction of several plant species, as plant metabolism and germination are greatly affected by temperature. Melanoxylon brauna, a tree species native to the Atlantic Forest that occurs from northeastern to southeastern Brazil, is one of the many species threatened by global warming. Despite the economic and ecological importance of M. brauna, studies investigating the influence of heat stress on seed germination and biochemical responses are still incipient. This study aimed to evaluate enzyme activity in the micropylar region of M. brauna seeds during germination under heat stress conditions. Endo-β-mannanase, α-galactosidase, polygalacturonase, pectin methylesterase, pectin lyase, total cellulase, 1,3-β-glucosidase, and 1,4-β-glucosidase activities were determined in micropyles of seeds imbibed for 24, 48 and 72 h at 25, 35 and 45 °C. Seed germination was highest at 25 °C. Endo-β-mannanase activity was not detected under any of the experimental conditions, but imbibition temperature had a significant effect on the activity of all other enzymes.

Index terms:
cell wall; enzyme; germination; temperature

Resumo:

Estudos recentes indicam alta na temperatura global no decorrer desse século, fato que pode ocasionar a extinção de diversas espécies vegetais, visto que processos como a germinação são influenciados pela temperatura. Dentre as espécies com risco de extinção está a Melanoxylon brauna, árvore nativa da Mata Atlântica de ocorrência nas regiões Nordeste e Sudeste. Mesmo diante de sua importância econômica e ecológica, estudos referentes à germinação em condições de estresse térmico, bem como suas consequências bioquímicas nas diferentes partes das sementes, ainda são incipientes. Diante disso, objetivou-se avaliar a germinação e a atividade enzimática na região micropilar de sementes de M. brauna durante a germinação sob estresse térmico. Avaliou-se germinação nas temperaturas de 25, 35 e 45 °C. As atividades das enzimas endo-β-mannanase, α- galactosidase, polygalacturonase, pectinametilesterase, pectinaliase, celulases totais, β-1,3- e β-1,4-glucosidases foram avaliadas em micrópilas embebidas por 0, 24, 48 e 72 h a 25, 35 e 45 °C. Houve maior germinação a 25 °C. Não foi detectada atividade da enzima endo-β-mananase em nenhuma das condições avaliadas. A temperatura de embebição influenciou a atividade das demais enzimas.

Termos para indexação:
parede celular; enzima; germinação; temperatura

INTRODUCTION

Melanoxylon brauna Schott is a tree native to the Brazilian Atlantic Forest. It occurs mainly in the southeast and northeast regions of the country. The tree has excellent wood properties, and its bark and sap are widely used in traditional medicine (Lorenzi, 2008LORENZI, H. Árvores brasileiras: manual de identificação e cultivo de plantas arbóreas nativas do Brasil. Nova Odessa: Instituto Platarum, 2008. ). However, because of the overexploitation of its timber, the species is currently classified as vulnerable in the official list of endangered flora of Brazil (Brasil, 2014BRASIL. Ministério do Meio Ambiente. Instrução Normativa nº 443, de 18 de setembro de 2014. Lista oficial das espécies da flora brasileira ameaçadas de extinção. <https://www.mma.gov.br/biodiversidade/conservacao-de-especies/fauna-ameacada/flora.html>.
https://www.mma.gov.br/biodiversidade/co...
). Information on seed germination may contribute to the development of conservation strategies for M. brauna.

A factor that may greatly affect the conservation status of M. brauna is climate change. Studies showed that global temperatures will rise by 1 to 5 °C over this century (PBMC, 2013PBMC. Painel Brasileiro de Mudanças Climáticas. Contribuição do grupo de trabalho 2 ao primeiro relatório de avaliação nacional do painel brasileiro de mudanças climáticas: sumário executivo. Rio de Janeiro, RJ, Brasil: PBMC, 2013. 28p. http://www.pbmc.coppe.ufrj.br/documentos/MCTI_PBMC_sumario_executivo_impactos_vulnerabilidades_e_adaptacao_WEB_3.pdf
http://www.pbmc.coppe.ufrj.br/documentos...
). Will the species be able to adapt to the effects of global warming? What can be done to prevent M. brauna and other vulnerable tree species from becoming extinct? To answer these questions, it is fundamental to increase the knowledge about the influence of temperature on the different physiological processes involved in seed germination.

Germination is mediated by a series of complex physical, physiological and biochemical processes. To emerge, the radicle must pass through the micropyle. Radicle emergence is preceded by the weakening of the micropylar endosperm and elongation of the embryonic axis (Yan et al., 2014YAN, D.; DUERMEYER, L.; LEOVEANU, C.; NAMBARA, E. The functions of the endosperm during seed germination. Plant and Cell Physiology, v.55, n.9, p.1521-1533, 2014. https://doi.org/10.1093/pcp/pcu089
https://doi.org/10.1093/pcp/pcu089...
). Although an essential part of seed germination, the mechanisms involved in the rupture of the micropylar endosperm, especially tissue weakening, are still little understood (Nonogaki et al., 2010NONOGAKI, H.; BASSEL, G.W.; BEWLEY, J.D. Germination: still a mystery. Plant Science, v.179, n.6, p.574-581, 2010. https://doi.org/10.1016/j.plantsci.2010.02.010
https://doi.org/10.1016/j.plantsci.2010....
). Such process is known to involve the loss of cell wall integrity by the action of hydrolases, transglycosylases, cellulases, hemicellulases, and reactive oxygen species (Borges et al., 2015BORGES, E.E.L.; ATAÍDE, G.M.; MATOS, A.C.B. Micropilar and embryonic events during hydration of Melanoxylon brauna Schott seeds. Journal of Seed Science, v.37, n.3, p.192-201, 2015. http://dx.doi.org/10.1590/2317-1545v37n3147846
http://dx.doi.org/10.1590/2317-1545v37n3...
; Koen et al., 2017KOEN, J.; SLABBERT, M.M.; BESTER, C.; BIERMAN, F. Germination characteristics of dimorphic honeybush (Cyclopia spp.) seed. South African Journal of Botany, v.110, p.68-74, 2017. https://doi.org/10.1016/j.sajb.2016.03.006
https://doi.org/10.1016/j.sajb.2016.03.0...
; Singh et al., 2017SINGH, V.P.; SINGH, S.; TRIPATHI, D.K.; PRASAD, S.M.; CHAUHAN, D.K. Reactive oxygen species in plants. John Wiley & Sons: Nova Jersey, 2017.). Enzymes activated at the initial stages of imbibition, such as β-mannanase, α-galactosidase (α-Gal), polygalacturonase (PG), pectin methylesterase (PME), pectin lyase (PL) and cellulase, are responsible for the degradation of cell wall polysaccharides and, consequently, the weakening of the micropylar endosperm, contributing to radicle emergence (Betts et al., 2017BETTS, N.S.; WILKINSON, L.G.; KHOR, S.F.; SHIRLEY, N.J.; LOK, F.; SKADHAUGE, B. Morphology, carbohydrate distribution, gene expression and enzymatic activities related to cell wall hydrolysis in four barley varieties during simulated malting. Frontiers in Plant Science, v.8, n.1872, 2017. https://www.frontiersin.org/articles/10.3389/fpls.2017.01872/full
https://www.frontiersin.org/articles/10....
; Mascher et al., 2017MASCHER, M.; GUNDLACH, H.; HIMMELBACH, A.; BEIER, S.; TWARDZIOK, S.; WICKER, T. A chromosome conformation capture ordered sequence of the barley genome. Nature, v.544, p.427-433, 2017. https://www.nature.com/articles/nature22043
https://www.nature.com/articles/nature22...
).

Temperature is a major factor influencing enzyme activity during germination. It can decrease, enhance, or even inhibit enzymatic processes and, therefore, accelerate or slow down seed metabolism (Laghmouchi et al., 2017LAGHMOUCHI, Y.; BELMEHDI, O.; BOUYAHYA, A.; SENHAJI, N.S.; ABRIN, J. Effect of temperature, salt stress and pH on seed germination of medicinal plant Origanum compactum. Biocatalysis and Agricultural Biotechnology, v.10, p.156-160, 2017. https://doi.org/10.1016/j.bcab.2017.03.002
https://doi.org/10.1016/j.bcab.2017.03.0...
). It may also influence the imbibition rate, altering the speed of chemical reactions that promote the mobilization of reserves and synthesis of necessary compounds for seedling growth. Thus, temperature is a determining factor for the occurrence of a species at a given locality (Medina et al., 2016MEDINA, C.V.; RODRIGUEZ, E.A.G.; BAGANTIM, A.G.; ANDRADE, R.A. Temperatura e armazenamento na germinação de sementes de Mabolo (Diospyros blancoi Willd). Nucleus, v.13, n.1, 2016. http://dx.doi.org/10.3738/1982.2278.1538
http://dx.doi.org/10.3738/1982.2278.1538...
).

Considering the importance of M. brauna, the predictions of climate change, the influence of temperature on germination, and the lack of information about biochemical processes occurring in the micropylar region, this study aimed to assess the activity of endo-β-mannanase, α-Gal, PG, PME, PL, total cellulase, 1,3-β-glucosidase and 1,4-β-glucosidase in the micropylar endosperm of M. brauna seeds during germination under heat stress conditions.

MATERIAL AND METHODS

Sample harvesting and preparation

Fruits of M. brauna were harvested in Leopoldina (21°31’55”S 42°38’35”W), Minas Gerais, southeastern Brazil, in September 2015. The pods were sun dried, manually threshed, and the seeds cleaned. Empty or damaged seeds and debris were discarded. Healthy, intact seeds were selected and stored in a cold chamber at 5 °C and 60% relative humidity until use. The experiments were conducted between February and August 2016. A completely randomized design with five replications of twenty seeds per treatment was used.

Germination test

Seeds were placed between two sheets of germination paper in petri dishes, moistened with distilled water, and incubated for 24, 48 and 72 h at 25, 35 or 45 °C in BOD incubators. Seeds were considered germinated upon radicle emergence.

Determination of enzyme activity

Enzyme activity was determined in the micropylar region of dry seeds and seeds imbibed for 24, 48 and 72 h at 25, 35 and 45 °C. Micropyles were extracted (Figure 1), subjected to the same conditions as described above, and analyzed for enzyme activity.

Figure 1
Representative photograph of a Melanoxylon brauna seed (A) and its micropylar region (B).

Endo-β-mannanase activity was determined by the gel-diffusion assay of Downie et al. (1994DOWNIE, B.; HILHORST, H.; BEWLEY, J.D. A new assay for quantifying endo-β-mananase activity using congo red dye. Phytochemistry, v.36, n.4, p.829-835, 1994. https://www.sciencedirect.com/science/article/pii/S0031942200904461
https://www.sciencedirect.com/science/ar...
), with modifications. The gel was first washed with distilled water, then incubated with buffer solution for thirty minutes, and washed once more with distilled water. Congo red dye (0.5% w/v) was added, the gel was incubated for thirty minutes, washed with ethanol for ten minutes, and rinsed with distilled water. NaCl solution (1 M) was added until a white halo was observed where the samples were pipetted.

α-Gal activity was quantified according to Borges et al., 2004BORGES, E.E.L.; REZENDE, S.T.; BORGES, R.C.G.; PEREZ, S.C.J.G.A. Caracterização de alfa-galactosidase em embrião e cotilédones de sementes de Platymiscium pubescens Micheli, var. pubecens (tamboril-da-mata).Revista Brasileira de Sementes, v.26, n.2, 2004.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0101-31222004000200012&lng=pt&tlng=pt
http://www.scielo.br/scielo.php?script=s...
. One unit of enzyme activity was defined as the amount of protein that releases 1 nmol of p-nitrophenol per minute under the assay conditions.

PG activity was determined by the 3,5-dinitrosalicylic acid (DNS) method as adapted by Miller (1959MILLER G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, v.31, n.3, p.426-428, 1959. https://pubs.acs.org/doi/abs/10.1021/ac60147a030
https://pubs.acs.org/doi/abs/10.1021/ac6...
). One unit of PG activity was defined as the amount of protein that produces 1 μmol of galacturonic acid per minute of reaction.

PME was extracted according to Pinto et al. (2011PINTO, L.K.A.; MARTINS, M.L.L.; RESENDE, E.D.; THIEBAUT, J.T.L. Atividade de pectina metilesterase e da β-galactosidase durante o amadurecimento do mamão cv. Golden. Revista Brasileira de Fruticultura, v.33, n.3, p.713-722, 2011. http://www.scielo.br/scielo.php?pid=S0100-29452011000300004&script=sci_abstract&tlng=pt
http://www.scielo.br/scielo.php?pid=S010...
), and its activity was quantified according to Grsic-Rausch and Rausch (2004). One unit of PME activity is equivalent to the amount of enzyme required to produce 1 μmol of NADPH per minute of reaction at 25 °C and pH 7.5.

PL activity was determined by the spectrophotometric method of Albersheim and Kilias (1962ALBERSHEIM, P.; KILIAS, U. Studies relating to the purification and properties of pectin transeliminase. Archives of Biochemistry and Biophysics, v.97, n.1, p.107-115, 1962. https://www.sciencedirect.com/science/article/pii/0003986162900504
https://www.sciencedirect.com/science/ar...
). Absorbance was read at 235 nm. Enzyme concentration was calculated using a molar absorption coefficient of 5550 L mol−1.cm−1 (Albersheim et al., 1996ALBERSHEIM, P.; DARVILL, A.G.; O’NEILL, M.A.; SCHOLS, H.A.; VORAGEN, A.G.J. An hypothesis: the same six polysaccharides are components of the primary cell walls of all higher plants. Progress in Biotechnology, v.14, p.47-55, 1996. https://doi.org/10.1016/S0921-0423(96)80245-0
https://doi.org/10.1016/S0921-0423(96)80...
).

Total cellulase activity was measured by the filter paper assay (Ghose, 1987GHOSE, T.K. Measurement of cellulase activities. Pure and Applied Chemistry, v.59, n.2, p.257-268, 1987. https://doi.org/10.1351/pac198759020257
https://doi.org/10.1351/pac198759020257...
). A 6 cm2 strip of filter paper was placed in a test tube containing 0.5 mL of sample (seed incubation solution) and 1.0 mL of 50 mM sodium acetate buffer pH 5.0. The reaction was interrupted by the addition of 1 mL of DNS. The concentration of reducing sugars was determined spectrophotometrically at 540 nm.

1,3-β-Glucosidase activity was determined using p-nitrophenyl β-d-glucopyranoside as substrate, and 1,4-β-glucosidase activity was determined using carboxymethyl cellulose as substrate. Reactions were conducted in 50 mM phosphate-buffered saline pH 6.0 (Singhania et al., 2013SINGHANIA, R.R.; PATEL, A.K.; SUKUMARAN, R.K.; LARROCHE, C.; PANDEY, A. Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresource Technology, v.127, p.500-507, 2013. https://doi.org/10.1016/j.biortech.2012.09.012
https://doi.org/10.1016/j.biortech.2012....
). Enzyme activity was expressed as units per gram of substrate.

Proteins were quantified by the Bradford method (1976BRADFORD, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, v.72, n.1-2, p.248-254, 1976. https://doi.org/10.1016/0003-2697(76)90527-3
https://doi.org/10.1016/0003-2697(76)905...
) using a standard curve (2.5-50 µg) of bovine serum albumin.

Statistical analysis

Data were subjected to analysis of variance followed by Tukey’s test at p < 0.05. Relationships between enzyme activity and germination conditions were investigated by regression analysis. Statistical analyses were performed using SAS version 9.2 (SAS Institute Inc., Cary, NC, USA).

RESULTS AND DISCUSSION

Radicle emergence was observed in seeds imbibed for 72 h. Germination was highest (83%) at 25 °C, decreasing to about 55% at 35 °C. Imbibition for 72 h at 45 °C led to complete loss of seed viability (Table 1). Temperature exerts a great influence on germination, as it regulates water absorption and biochemical reactions involved in seed metabolism (Bewley et al., 2013BEWLEY, J.D.; BRADFORD, K.J.; HILHORST, H.W.M.; NONOGAKI, H. Seeds: physiology of development, germination and dormancy. New York: Springer, 2013. ; Graeber et al., 2014GRAEBER, K.; LINKIES, A.; STEINBRECHER, T.; MUMMENHOFF, K.; TARKOWSKA, D.; TURECKOVA, V.; IGNATZ, M.; SPERBER, K.; VEOGELE, A.; JONG; H.; URBANOVÁ, T.; STRNAD, M.; LEUBNER-METZGER, G. Delay of germination mediates a conserved coat-dormancy mechanism for the temperature- and gibberellin-dependent control of seed germination. Proceedings of the National Academy of Sciences, v.111, n.34, p.3571-3580, 2014. https://doi.org/10.1073/pnas.1403851111
https://doi.org/10.1073/pnas.1403851111...
).

Table 1
Germination percentage of Melanoxylon brauna seeds at different temperatures.

In general, seeds of subtropical and tropical species germinate at 20-30 °C (Oliveira et al., 2016OLIVEIRA, F.N.; FRANÇA, F.D.; TORRES, S.B.; NOGUEIRA, N.W.; FREITAS, R.M.O. Temperaturas e substratos na germinação de sementes de pereiro-vermelho (Simira gardneriana M.R. Barbosa & Peixoto). Revista Ciência Agronômica, v.47, n.4, p.658-666, 2016. http://dx.doi.org/10.5935/1806-6690.20160079
http://dx.doi.org/10.5935/1806-6690.2016...
; Silva et al., 2016SILVA, M.L.M.; ALVES, E.U.; BRUNO, R.L.L.; SANTOS-MOURA, S.S.; SANTOS-NETO, A.P. Germinação de sementes de Chorisia glaziovii O. Kuntze submetidas ao estresse hídrico sob diferentes temperaturas. Ciência Florestal , v.26, n.3, p.999-1007, 2016. https://www.redalyc.org/pdf/534/53447685028.pdf
https://www.redalyc.org/pdf/534/53447685...
). The germination behavior of M. brauna seeds observed in the current study agrees with that reported in the literature. Flores et al. (2014FLORES, A.V.; BORGES, E.E.L.; GUIMARÃES, V.M.; ATAÍDE, G.M.; CASTRO, R.V.O. Germinação de sementes de Melanoxylon brauna Schott em diferentes temperaturas. Revista Árvore, v.38, n.6, p.1147-1154. 2014. http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-67622014000600019
http://www.scielo.br/scielo.php?script=s...
) found that M. brauna seeds were able to germinate between 12.3 and 42.5 °C, but not at 45 °C; the optimum temperature was 27 °C. Imbibition at 45 °C for 72 h was shown to cause irreversible damage to M. brauna seeds, impairing their germination, even when seeds were later transferred to 25 °C (Santos et al., 2017SANTOS, M.M.; BORGES, E.E.L.; ATAÍDE, M.M.; SOUZA, G.A. Germination of seeds of Melanoxylon brauna Schott. under heat stress: production of reactive oxygen species and antioxidant activity. Forests, v.8, n.11, p.405, 2017. https://www.mdpi.com/1999-4907/8/11/405
https://www.mdpi.com/1999-4907/8/11/405...
). Radicle emergence depends not only on the growth potential of the embryo, but also on the reduction in mechanical resistance in the micropylar region (Bewley et al., 2013BEWLEY, J.D.; BRADFORD, K.J.; HILHORST, H.W.M.; NONOGAKI, H. Seeds: physiology of development, germination and dormancy. New York: Springer, 2013. ). Although the physical and biochemical aspects involved in this process are not fully understood, it is known that hydrogen peroxide production, hydrolases, and cellulases have an important role in weakening the cell wall (Zhang et al., 2014ZHANG, Y.; CHEN, B.; XU, Z.; SHI, Z.; CHEN, S.; HUANG, X.; CHEN, J.; WANG, X.F. Involvement of reactive oxygen species in endosperm cap weakening and embryo elongation growth during lettuce seed germination. Journal of Experimental Botany, v.65, n.12, p.3189-3200, 2014. https://doi.org/10.1093/jxb/eru167
https://doi.org/10.1093/jxb/eru167...
; Santos et al., 2017SANTOS, M.M.; BORGES, E.E.L.; ATAÍDE, M.M.; SOUZA, G.A. Germination of seeds of Melanoxylon brauna Schott. under heat stress: production of reactive oxygen species and antioxidant activity. Forests, v.8, n.11, p.405, 2017. https://www.mdpi.com/1999-4907/8/11/405
https://www.mdpi.com/1999-4907/8/11/405...
).

Enzyme activity was not observed in isolated micropyles imbibed for up to 72 h. These results suggest that hydrolases and cellulases are produced in the embryonic axis and transferred to the micropylar region during germination, which reinforces the hypothesis that these enzymes contribute to radicle emergence by weakening the micropylar region.

Endo-β-mannanase activity was not detected under any of the evaluated conditions. Previous reports showed that its activity is intensified at the end of the germination process, mainly during seedling formation (Ferreira et al., 2018FERREIRA, V.F.; RICALDONI, M.A.; ROSA, S.V.B.; FANTASINI, T.B. Endo-β-mannanase enzyme activity in the structures of Coffea arabica L. seeds under different types of processing and drying.Ciência Rural, v.48, n.12, 2018. https://dx.doi.org/10.1590/0103-8478cr20170839
https://dx.doi.org/10.1590/0103-8478cr20...
). The activity of all other enzymes differed significantly with temperature. α-Gal activity was highest at 25 and 35 °C (Figure 2A). PG, PME and PL showed higher activities after 72 h of imbibition at 45 °C (Figures 2B, 2C and 2D). These enzymes are crucial for cell wall degradation and radicle emergence (Borges et al., 2015BORGES, E.E.L.; ATAÍDE, G.M.; MATOS, A.C.B. Micropilar and embryonic events during hydration of Melanoxylon brauna Schott seeds. Journal of Seed Science, v.37, n.3, p.192-201, 2015. http://dx.doi.org/10.1590/2317-1545v37n3147846
http://dx.doi.org/10.1590/2317-1545v37n3...
; Bicalho et al., 2016BICALHO, E.M.; MOTOIKE, S.Y.; BORGES, E.E.L.; ATAÍDE, G.M.; GUIMARÃES, V.M. Enzyme activity and reserve mobilization during Macaw palm (Acrocomia aculeata) seed germination. Acta Botanica Brasilica, v.30, n.3, p.438-444, 2016. http://dx.doi.org/10.1590/0102-33062016abb0181
http://dx.doi.org/10.1590/0102-33062016a...
).

Figure 2
α-Galactosidase (A), polygalacturonase (B), pectin methylesterase (C) and pectin lyase (D) activities in the micropylar region of Melanoxylon brauna seeds during imbibition at 25, 35 and 45 °C.

α-Gal activity depends on environmental conditions, including temperature (Coffigniez et al., 2018COFFIGNIEZ, F.; BRIFFAZ, A.; MESTRES, C.; RICCI, J.; ALTER, P.; DURAND, N.; BOHUON, P. Kinetic study of enzymatic α-galactoside hydrolysis in cowpea seeds. Food Research International, v.113, p.443-451, 2018. DOI10.1016/j.foodres.2018.07.030 ). In M. brauna seeds, the highest activity occurred at the optimal germination temperature (25 °C). In addition to metabolizing carbohydrate reserves, α-Gal hydrolyzes cell wall polysaccharides and raffinose family oligosaccharides, thereby providing energy for germination (Bicalho et al., 2016BICALHO, E.M.; MOTOIKE, S.Y.; BORGES, E.E.L.; ATAÍDE, G.M.; GUIMARÃES, V.M. Enzyme activity and reserve mobilization during Macaw palm (Acrocomia aculeata) seed germination. Acta Botanica Brasilica, v.30, n.3, p.438-444, 2016. http://dx.doi.org/10.1590/0102-33062016abb0181
http://dx.doi.org/10.1590/0102-33062016a...
; Farias et al., 2015FARIAS, E.T; SILVA, E.A.A.; TOOROP, P.E.; BEWLEY, J.D.; HILHORST, H.M. Expression studies in the embryo and in the micropylar endosperm of germinating coffee (Coffea arabica cv. Rubi) seeds. Plant Growth Regulation, v.75, n.2, p.575-581, 2015. https://link.springer.com/article/10.1007/s10725-014-9960-6
https://link.springer.com/article/10.100...
). The low enzyme activity found at 45 °C shows that α-Gal is sensitive to heat stress. Similar to the observed in the current study, α-Gal activity was highest at 25 °C in Dalbergia nigra seeds (Ataíde et al., 2016ATAÍDE, G.M.; BORGES, E.E.L.; GONÇALVES, J.F.C.; GUIMARÃES, V.M.; FLORES, A.V. Alterações fisiológicas durante a hidratação de sementes de Dalbergia nigra ((Vell.) Fr. All. ex Benth.). Ciência Florestal, v.26, n.2, p.615-625, 2016. http://www.redalyc.org/articulo.oa?id=53446151024
http://www.redalyc.org/articulo.oa?id=53...
).

PG is essential for the germination of Schizolobium parahyba and Arabidopsis sp. seeds (Magalhães et al., 2009MAGALHÃES, S.M.; BORGES, E.E.L.; BERGER, A.P. Alterações nas atividades das enzimas alfa-galactosidade e poligalacturonase e nas reservas de carboidratos de sementes de Schizolobium parahyba (vell.) Blake (guapuruvú) durante a germinação. Revista Brasileira de Sementes , v.31, n.2, p.253-261, 2009. http://www.scielo.br/scielo.php?pid=S0101-31222009000200030&script=sci_abstract&tlng=pt
http://www.scielo.br/scielo.php?pid=S010...
; Han and Yang, 2015HAN, C.; YANG, P. Studies on the molecular mechanisms of seed germination. Proteomics, v.15, p.1671-1679, 2015. https://onlinelibrary.wiley.com/doi/epdf/10.1002/pmic.201400375
https://onlinelibrary.wiley.com/doi/epdf...
; Scheler et al., 2015SCHELER, C.; WEITBRECTHT, K.; PEARCE, S.P.; HAMPSTEAD, A.; BUTTNER-MAINIK, A.; LEE, K.J.D.; VOEGELE, A.; ORACZ, K.; BAS, J.W.; WANG, X.F.; WOOD, A.T.A.; BENTSINK, L.; KING, J.R.; KNOX, P.; HOLDSWORTH, M.J.; MULLER, K.; LEUBNER-METZGER, G. Promotion of testa rupture during garden cress germination involves seed compartment-specific expression and activity of pectin methylesterase. Plant Physiology, v.167, p.200-215, 2015. http://www.plantphysiol.org/content/plantphysiol/167/1/200.full.pdf
http://www.plantphysiol.org/content/plan...
). The enzyme catalyzes the hydrolysis of 1,4-α-glycosidic bonds between galacturonic acid residues in the pectin chain. In the present study, PG activity was higher at 45 °C for all imbibition times. The optimum germination temperature does not always coincide with the optimum temperature for enzyme activity. In D. nigra seeds, PG activity peaked at 40, 45 and 60 °C, but germination percentage was highest at 25 °C (Ataíde et al., 2016ATAÍDE, G.M.; BORGES, E.E.L.; GONÇALVES, J.F.C.; GUIMARÃES, V.M.; FLORES, A.V. Alterações fisiológicas durante a hidratação de sementes de Dalbergia nigra ((Vell.) Fr. All. ex Benth.). Ciência Florestal, v.26, n.2, p.615-625, 2016. http://www.redalyc.org/articulo.oa?id=53446151024
http://www.redalyc.org/articulo.oa?id=53...
). In fruits of Uapaca kirkiana, Ziziphus mauritiana, Tamarindus indica and Berchemia, the optimum temperature range for PG activity was shown to be 25 to 37 °C (Muchuweti et al., 2005MUCHUWETI, M.; MOYO, E.; MUSHIPE, S. Some properties of the polygalacturonase from four Zimbabwean wild fruits (Uapaca kirkiana, Ziziphus mauritiana, Tamarindus indica and Berchemia discolor fruits). Food Chemistry, v.90, n.4, p.655-661, 2005. https://www.sciencedirect.com/science/article/pii/S0308814604003784?via%3Dihub
https://www.sciencedirect.com/science/ar...
). In Prunus persica fruits, PG activity was highest during imbibition at 35 °C (Sainz and Vendrusculo, 2015SAINZ, R.L.; VENDRUSCULO, J.L.S. Propriedade da poligalacturonase e pectinametilesterase em pêssego (Prunus persica (L.) Batsch) de cultivares brasileiras. Revista Brasileira de Tecnologia Agroindustrial, v.9, n.1, p.1724-1743, 2015. https://periodicos.utfpr.edu.br/rbta/article/view/2403
https://periodicos.utfpr.edu.br/rbta/art...
).

PME and PG have related functions. PME catalyzes the de-esterification of pectic substances by hydrolyzing methyl ester groups, producing pectin with a lower degree of methylation, which is then used as a substrate by PG (Sainz and Vendrusculo, 2015SAINZ, R.L.; VENDRUSCULO, J.L.S. Propriedade da poligalacturonase e pectinametilesterase em pêssego (Prunus persica (L.) Batsch) de cultivares brasileiras. Revista Brasileira de Tecnologia Agroindustrial, v.9, n.1, p.1724-1743, 2015. https://periodicos.utfpr.edu.br/rbta/article/view/2403
https://periodicos.utfpr.edu.br/rbta/art...
). PME activity increased after 24 h of imbibition at 25, 35 and 45 °C, but decreased after 48 h at 35 °C. At the basis of these results, it is possible to infer that the enzyme is produced before germination and is associated with the weakening of the seed coat and the degradation of the micropylar endosperm. These results agree with those obtained by Borges et al. (2015BORGES, E.E.L.; ATAÍDE, G.M.; MATOS, A.C.B. Micropilar and embryonic events during hydration of Melanoxylon brauna Schott seeds. Journal of Seed Science, v.37, n.3, p.192-201, 2015. http://dx.doi.org/10.1590/2317-1545v37n3147846
http://dx.doi.org/10.1590/2317-1545v37n3...
). The authors reported an increase in PME activity during imbibition of M. brauna seeds at 30 °C. PME activity was detected in Lepidium sativum seeds, suggesting that the enzyme plays an important role in testa rupture during radicle emergence (Scheler et al., 2015SCHELER, C.; WEITBRECTHT, K.; PEARCE, S.P.; HAMPSTEAD, A.; BUTTNER-MAINIK, A.; LEE, K.J.D.; VOEGELE, A.; ORACZ, K.; BAS, J.W.; WANG, X.F.; WOOD, A.T.A.; BENTSINK, L.; KING, J.R.; KNOX, P.; HOLDSWORTH, M.J.; MULLER, K.; LEUBNER-METZGER, G. Promotion of testa rupture during garden cress germination involves seed compartment-specific expression and activity of pectin methylesterase. Plant Physiology, v.167, p.200-215, 2015. http://www.plantphysiol.org/content/plantphysiol/167/1/200.full.pdf
http://www.plantphysiol.org/content/plan...
).

PL activity was highest at 45 °C and increased with imbibition time at all temperatures. PL breaks down oligogalacturonides of the cell wall, deteriorating the lateral endosperm. The enzyme also induces the synthesis of expansins, which are mediators of the germination process (Zhao et al., 2008ZHAO, Q.; YUAN, S.; WANG, X.; ZHANG, Y.; ZHU, H. Restoration of mature etiolated cucumber hypocotyl cell wall susceptibility to expansin by pretreatment with fungal pectinases and EGTA in vitro. Plant Physiology , v.147, p.1874-1885, 2008. http://www.plantphysiol.org/content/plantphysiol/147/4/1874.full.pdf
http://www.plantphysiol.org/content/plan...
; Cao, 2012CAO, J. The pectin lyases in Arabidopsis thaliana: evolution, selection and expression profiles. Plos One, v.7, n.10, 2012. https://doi.org/10.1371/journal.pone.0046944
https://doi.org/10.1371/journal.pone.004...
; Sainz and Vendrusculo, 2015SAINZ, R.L.; VENDRUSCULO, J.L.S. Propriedade da poligalacturonase e pectinametilesterase em pêssego (Prunus persica (L.) Batsch) de cultivares brasileiras. Revista Brasileira de Tecnologia Agroindustrial, v.9, n.1, p.1724-1743, 2015. https://periodicos.utfpr.edu.br/rbta/article/view/2403
https://periodicos.utfpr.edu.br/rbta/art...
).

Although hydrolases play a fundamental role in M. brauna seed germination, under heat stress (45 °C), high PG, PME and PL activities may contribute to seed deterioration. It is possible that an excessive increase in enzyme activity enhanced reserve degradation, accelerating the loss of cell wall integrity and increasing damage to cell membranes (Santos et al., 2017SANTOS, M.M.; BORGES, E.E.L.; ATAÍDE, M.M.; SOUZA, G.A. Germination of seeds of Melanoxylon brauna Schott. under heat stress: production of reactive oxygen species and antioxidant activity. Forests, v.8, n.11, p.405, 2017. https://www.mdpi.com/1999-4907/8/11/405
https://www.mdpi.com/1999-4907/8/11/405...
).

Total cellulase and 1,4-β-glucosidase activities were highest at 35 °C after 48 h of imbibition (Figures 3A and 3C). This temperature also favored 1,3-β-glucosidase activity, which was found to increase with imbibition time (Figure 3B). At 45 °C, 1,3-β- and 1,4-β-glucosidase activities were lowest after 72 h of imbibition. This result is likely due to the loss of seed vigor caused by heat stress, resulting in protein denaturation and loss of enzyme activity (Santos et al., 2017SANTOS, M.M.; BORGES, E.E.L.; ATAÍDE, M.M.; SOUZA, G.A. Germination of seeds of Melanoxylon brauna Schott. under heat stress: production of reactive oxygen species and antioxidant activity. Forests, v.8, n.11, p.405, 2017. https://www.mdpi.com/1999-4907/8/11/405
https://www.mdpi.com/1999-4907/8/11/405...
). Cellulases are responsible for the degradation of cellulose, a major component of the plant cell wall. β-Glucosidases break the chemical bond between the glucose units of cellobiose, releasing free glucose. By doing so, they contribute to the weakening of the micropylar endosperm and provide energy for radicle emergence, as observed in seeds of Coffea arabica (Castro and Pereira, 2010CASTRO, A.M.; PEREIRA, J.R.N. Produção, propriedades e aplicação de celulases na hidrólise de resíduos agroindustriais. Química Nova, v.33, n.1, p.181-188, 2010. http://submission.quimicanova.sbq.org.br/qn/qnol/2010/vol33n1/30-RV09352.pdf
http://submission.quimicanova.sbq.org.br...
), Lactuca sativa (Chen et al., 2016CHEN, B.; MA, J.; XU, Z.; WANG, X. Abscisic acid and ethephon regulation of cellulase in the endosperm cap and radicle during lettuce seed germination. Journal of Integrative Plant Biology, v.58, n.10, p.859-869, 2016. https://onlinelibrary.wiley.com/doi/epdf/10.1111/jipb.12479
https://onlinelibrary.wiley.com/doi/epdf...
) and L. sativum (Ogórek, 2016OGÓREK, R. Enzymatic activity of potential fungal plant pathogens and the effect of their culture filtrates on seed germination and seedling growth of garden cress (Lepidium sativum L.). European Journal of Plant Pathology, v.145, n.2, p.469-481, 2016. https://link.springer.com/article/10.1007/s10658-016-0860-7
https://link.springer.com/article/10.100...
).

Figure 3
Total cellulase (A), 1,3-β-glucosidase (B) and 1,4-β-glucosidase (C) activities in the micropylar region of Melanoxylon brauna seeds during imbibition at 25, 35 and 45 °C.

Enzyme activity was related to germination percentage, but was less affected by high temperatures. The increase in enzyme activity in heat-stressed seeds probably contributed to cell wall degradation, leading to the accumulation of reactive oxygen species and membrane damage (Santos et al., 2017SANTOS, M.M.; BORGES, E.E.L.; ATAÍDE, M.M.; SOUZA, G.A. Germination of seeds of Melanoxylon brauna Schott. under heat stress: production of reactive oxygen species and antioxidant activity. Forests, v.8, n.11, p.405, 2017. https://www.mdpi.com/1999-4907/8/11/405
https://www.mdpi.com/1999-4907/8/11/405...
). The results show that PG, PME, PL, total cellulase, 1,3-β-glucosidase and 1,4-β-glucosidase activities can be used to assess the physiological quality of M. brauna seeds.

CONCLUSIONS

M. brauna seeds showed optimal germination at 25 °C. Imbibition at 45 °C for 72 h resulted in the death of all seeds. Endo-β-mannanase activity was not detected after 72 h of imbibition at any of the tested temperatures. α-Gal activity was highest after 48 h of imbibition at 25 °C and lowest after 24 h at 45 °C. PG activity was highest after 48 h of imbibition at 45 °C and lowest after imbibition at 25 °C. PME and PL activities increased during 72 h of imbibition at 45 °C, but decreased during imbibition at 25 °C. Total cellulase, 1,3-β-glucosidase, and 1,4-β-glucosidase activities where highest during the first hours of imbibition at 45 °C, but decreased markedly after 48 h. High PG, PME, PL, total cellulase, 1,3-β-glucosidase and 1,4-β-glucosidase activities during imbibition indicate the occurrence of heat stress in M. brauna seeds.

ACKNOWLEDGMENTS

The authors thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Pró-Amazonas Project, nº 52), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for their financial support.

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Publication Dates

  • Publication in this collection
    23 Mar 2020
  • Date of issue
    2020

History

  • Received
    14 Oct 2019
  • Accepted
    10 Jan 2020
ABRATES - Associação Brasileira de Tecnologia de Sementes Av. Juscelino Kubitschek, 1400 - 3° Andar, sala 31 - Centro,, CEP 86020-000 Londrina/PR - Londrina - PR - Brazil
E-mail: jss@abrates.org.br