ANTIOXIDANT ACTIVITY AND PHYSIOLOGICAL EFFECTS IN dubium (Sprengel) Taubert AFTER OVERCOMING DORMANCY TREATMENTS WITH WATER IN DIFFERENT TEMPERATURES Maiara Iadwizak Ribeiro, Erly Carlos Porto, Evelin Maria Muller, Guilherme de Almeida Garcia Rodrigues, Henrique Roberto Gattermann Mittelstaedt, Andréa Maria Teixeira Fortes and Jaqueline Malagutti Corsato

SUMMARY

This study aimed to evaluate the physiological behavior, the average time, frequency and synchrony) were analyzed. The en- antioxidant enzymes activity and the lipoperoxide levels in Pelto- zymes catalase and peroxidase were quantified after 2, 4, 8, 12 phorum dubium (Sprengel) Taubert seeds when submitted to and 24h of imbibition. It is concluded that the best methods for different immersion times and water temperatures to overcome overcoming dormancy in P. dubium were the treatments us- physical dormancy. P. dubium seeds were submitted to treatments ing water at 75 and 95ºC because, due to the efficient action of with heated water (25, 50, 75 e 95ºC) during 24h, with and with- the enzymes catalase and peroxidase, the levels of lipid peroxida- out previous scarification, in order to overcome dormancy. Seed tion were controlled, allowing the seeds to achieve high germina- physiology, electrical conductivity and germination (percentage, tion and coefficient of velocity of germination.

Introduction system comprised by actions state (Pereira and Rodrigues, the use of native species that that try to reestablish the con- 2012). In order to achieve it, act creating a new vegetation The recovery of degraded ditions of the disturbed envi- one of the used methods is cover that is capable of sus- areas consists in a complex ronment, back to its natural re-vegetation, characterized by taining itself, thereby assisting

KEYWORDS / Catalase / Canafistula / Native Species / Peroxidase / Recovery of Degraded Areas / Received: 03/02/2017. Modified: 03/21/2018. Accepted: 03/28/2018.

Maiara Iadwizak Ribeiro. Bio- nagement of Natural Resour- , Universidade Federal Unioeste, Brazil. e-mail: an- logist, M.Sc. in Conservation ces, Unioeste, Brazil. e-mail: de Santa Catarina (UFSC), [email protected] and Management of Natural erly.carlos@ gmail.com Brazil. e-mail: guilhermegar- Jaqueline Malagutti Corsato. Resources, Universidade Esta- Evelin Maria Muller. Biologist, [email protected] Biologist, Unioeste, Brazil. dual do Oeste do Paraná M.Sc. in Conservation and Ma- Henrique Roberto Gattermann M.Sc. and Ph.D. in Biological (Unioeste), Brazil. Address: nagement of Natural Resour- Mittelstaedt. Biologist, Unio- Sciences (Botany), UNESP, Centro de Ciências Biológicas ces, Unioeste, Brazil. e-mail: este, Brazil. e-mail: henrique. Brazil. Professor, Unioeste, e da Saúde, Unioeste. Rua [email protected] [email protected] Brazil. e-mail: jaquecorsato@ Universitária, 1619, Universitá- Guilherme de Almeida Garcia Andréa Maria Teixeira Fortes. hotmail.com rio: CEP: 85819-110 - Cascavel Rodrigues. Biologist, M.Sc. in Biologist, M.Sc. and Ph.D. in PR Brazil. e-mail: maiara.iad- Conservation and Management Biological Sciences (Botany), wizak@ gmail.com of Natural Resources, Unio- Universidade Estadual Paulista Erly Carlos Porto. Biologist, este, Brazil. Ph.D candidate in Julio de Mesquita Filho M.Sc. in Conservation and Ma- Biology of Fungi, Algae and (UNESP), Brazil. Professor,

8 0378-1844/14/07/468-08 $ 3.00/0 JANUARY 2019 • VOL. 44 Nº 1 ACTIVIDAD ANTIOXIDANTE Y EFECTOS FISIOLÓGICOS EN SEMILLAS DE Peltophorum dubium (Sprengel) Taubert DESPUÉS DE TRATAMIENTOS DE SUPERACIÓN DE LA LATENCIA CON AGUA A DIFERENTES TEMPERATURAS Maiara Iadwizak Ribeiro, Erly Carlos Porto, Evelin Maria Muller, Guilherme de Almeida Garcia Rodrigues, Henrique Roberto Gattermann Mittelstaedt, Andréa Maria Teixeira Fortes y Jaqueline Malagutti Corsato

RESUMEN El objetivo fue evaluar el comportamiento fisiológico, la ac- índice, frecuencia y sincronización de la germinación. Las en- tividad de las enzimas antioxidantes y niveles de lipoperóxi- zimas catalasa y peroxidasa se cuantificaron después de 2, 4, dos en las semillas de Peltophorum dubium (Sprengel) Taubert 8, 12 y 24h de imbibición. Se concluye que los mejores mé- cuando se someten a inmersión en agua caliente para superar todos para romper la latencia en P. dubium fueron los trata- la latencia física. Semillas de P. dubium fueron sometidas a mientos con agua calentada a 75 y 95ºC, ya que debido a la tratamientos de superación de latencia con agua calentada a acción eficaz de las enzimas catalasa y peroxidasa, los niveles diferentes temperaturas (25, 50, 75 y 95ºC) durante 24h, con de peroxidación lipídica se controlaron lo que permite que las y sin escarificación previa. En la etapa fisiológica se evaluó semillas presentan elevada germinación e índice de velocidad la conductividad eléctrica, y el porcentaje, tiempo promedio, de germinación.

ATIVIDADE ANTIOXIDANTE E EFEITOS FISIOLÓGICOS EM SEMENTES DE Peltophorum dubium (Sprengel) Taubert APÓS TRATAMENTOS DE SUPERAÇÃO DE DORMÊNCIA COM ÁGUA EM DIFERENTES TEMPERATURAS Maiara Iadwizak Ribeiro, Erly Carlos Porto, Evelin Maria Muller, Guilherme de Almeida Garcia Rodrigues, Henrique Roberto Gattermann Mittelstaedt, Andréa Maria Teixeira Fortes e Jaqueline Malagutti Corsato RESUMO O objetivo foi avaliar o desempenho fisiológico, a ativida- frequência e sincronização da germinação. As enzimas catala- de das enzimas antioxidantes e os níveis de lipoperóxido nas se e peroxidase foram quantificadas após 2, 4, 8, 12 e 24h de sementes de Peltophorum dubium (Sprengel) Taubert quando embebição. Conclui-se que os melhores métodos para supera- submetidas a diferentes tempos de imersão e temperaturas de ção de dormência em P. dubium foram os tratamentos utilizan- água para superação de dormência física. As sementes de P. do água aquecida a 75 e 95ºC, pois devido a ação eficiente dubium foram submetidas a tratamentos de superação de dor- das enzimas catalase e peroxidase, os níveis de peroxidação mência com água aquecida (25, 50, 75 e 95ºC) por 24h, com e lipídica foram controlados permitindo assim que as sementes sem escarificação prévia. Na etapa fisiológica foi avaliada a apresentassem elevada germinação e índice de velocidade de condutividade elétrica, e a porcentagem, tempo médio, índice, germinação.

in the recovery of the present The use of heated water is a subjected to treatments for with alternate and compound vegetation (Guglieri-Caporal promising treatment for over- overcoming dormancy using semidecidous , that plays et al., 2011). coming dormancy, being a low heated water is being increas- the role of secondary species The production of native cost and effective approach for ingly related to mechanisms of in the ecological succession seedlings is an essential step to various native species (Galindo, membrane repair present in and has a wide distribution in the recovery of degraded areas. 2006). Its efficiency is verified cells, where it is possible the Parana territory (Duarte The production of these seed- through the softening of the rig- to verify the action of antioxi- and Krentkowski, 2014). lings requires studies about the id tegument that helps water dant enzymes as peroxidase, As most of , P. du- species to be used, such as bo- absorption, being effective in the catalase and superoxide dis- bium seeds have a thick and ri- tanical identification, seed pro- germination of several species. mutase. These enzymes remove gid tegument, an epidermis for- cessing and storage, and meth- However, for some species the and degrade the reactive oxy- med by macrosclereids, which ods for overcoming dormancy, continuous use of water heated gen species (ROS), since the are covered with a grease cuti- since approximately two thirds to high temperatures causes production of these free radi- cle and a layer of hemicellulose, of seeds of native tree species damage to the embryo, impeding cals causes stress, compromis- accompanied by a layer of os- used to recover degraded areas the occurrence of germination ing the integrity of the mem- teosclereids. These cells water- present dormancy (Santos et al., (Albulquerque et al., 2007; branes, cell metabolism and, proof the seed tegument, pre- 2011; Freire et al., 2016, Abreu Zucareli et al., 2010). Ribeiro consequently germination venting the gas exchange and et al., 2017). et al. (2016) reported that the (Nkang and Omokaro, 2000; water entrance that occurs The physical dormancy is the use of water at 95ºC in P. dubi- Ataide et al., 2016). during the germination, result- most common one, mainly in um seeds did not promote ger- Taking into account the use ing in dormant seeds (Souza, the Fabaceae family. In laborato- mination (0%), under the hy- of native species in the recov- 2009; Ribeiro et al., 2016). ry, methods are being tested that pothesis that in these conditions ery of degraded areas, the The present study aimed to facilitate to overcome dormancy, a denaturation of proteins and species Peltophorum dubium evaluate the physiological be- therefore enhancing the germina- enzymes occurred, causing dam- Spreng (Taubert), a member of havior, the activity of antioxi- tion percentage of these species, ages to the embryonic tissue. the Fabaceae family popularly dant enzymes and the levels allowing a high productivity in The good or bad perfor- known as canafistula, is high- of lipoperoxide in seeds of the nursery (Oliveira, 2013). mance of germination in seeds lighted. It is a mediumsized tree, Peltophorum dubium (Spren-

JANUARY 2019 • VOL. 44 Nº 1 9 gel) Taubert when subjected to with electrode of 1.0 constant. bance readings at 240nm were average it was observed that immersion in heated water in The results were expressed in measured using a spectropho- seeds that did not go through order to overcome physical µS·cm-1·g-1, according to the tometer. The coefficient of mo- the mechanical scarification dormancy. methodology described in lar extinction of H2O2 (39.4 presented smaller rates for this AOSA (1983) and Perez and mmol·l-1·cm-1) was calculated variable (~145,78µS·cm-1·g-1). Methodology Negreiros (2001). and the result was expressed in This occurs because when The germination test was mmol of consumed H2O2 per there is no mechanical scarifi- The experiment was carried carried out according to Brasil min-1·mg-1 of protein. cation of the seeds of P. du- out in the Plant Physiolo- (2009). The seeds were accom- The lipoperoxide was deter- bium, the imbibition is slower, gy Laboratory of the West modated in ‘Germitest’ paper mined according to Heath and as compared to seeds that went of Parana State University rolls, and all the treatments Packer (1968), cited by Rama through previous mechanic (Unioeste), Cascavel campus, stored in a B.O.D. germination Devi and Prasad (1998). scarification. Thus, there time from March to September of chamber, with constant tem- Solutions of tiobarbituric acid needed for the cell membranes 2016. The seeds of Pelto- perature of 25ºC and photope- (TBA) 0.25% and trichloroa- to go back to its crystalline phorum dubium were donated riod light:dark of 12:12h. cetic acid (TCA) 10% were state without cell damage, de- by the Itaipu seedling nursery, Germinated seeds counts used, with 100 mg of material. creasing lixiviation of solutes Foz do Iguaçu, and presented a were carried out daily until the The absorbance readings were (Ataide et al., 2016). 12% of water content. 14º day after the installation of measured using a spectropho- In seeds of Brachiaria bri- The seeds underwent an the test (Brasil, 2013). The tometer UV-visible at 560 and zantha that also show physical asepsis process according to seeds were considered germi- 600 nm. For the calculations, dormancy, Cardoso et al. (2014) Brasil (2013) and were subject- nated if they presented 2mm of the coefficient of molar ex- used the treatment of acid scar- ed to the treatments of dor- primary root length (Laboriau, tinction of malondialdehyde ification for 5min to overcome mancy overcoming, in a com- 1983). The analyzed parameters (155mmol·l-1·cm-1) was used, dormancy during the accelerated pletely randomized design with were: germination percentage being expressed in nmol of ageing, and similarly observed a factorial 2×4 scheme (factor (G), mean germination time tiobarbituric acid (TBARS) that those seeds led to a higher 1, seeds with and without pre- (MGT; Nakagawa, 1994), ger- per g of reactive substances of electrical conductivity during vious scarification by sandpa- mination speed index (GSI), fresh matter. the process, presenting a signif- per in the opposite side of the germination frequency and icant increase of 38.24µS·cm-1·g-1 micropyle × factor 2, four dif- synchronization (U), (Santana Statistical analysis compared to the control without ferent water temperatures for and Ranal, 2004). scarification. immersion during 24h). The The physiological data were It was verified that, on aver- different temperatures em- Specific antioxidant enzyme analyzed following normality age, for seeds of P. dubium the ployed were 25, 50, 75 and and lipoperoxide activity assumptions (Shapiro-Wilk) water temperature of 95ºC uti- 95ºC, totalizing 8 treatments. and homoscedasticity (Levene lized to overcome the dormancy The enzymatic tests were The peroxidase (POD) activ- Test). If p>0.5 they were sub- led to significantly greater elec- conducted utilizing seeds with ity in seeds of P. dubium treat- jected to the analysis of vari- trical conductivity than the oth- or without previous scarifica- ed with heated water to over- ance (ANOVA - F Test) and ers, with an increase of tion in a factorial 4×5 scheme, come dormancy, with and the averages compared by the 50,00µS·cm-1·g-1 in comparison with 25 sampling units in each without scarification, was de- Skott-Knott test with 5% prob- to 25ºC (Table I). Ataíde et al. replication. The 20 treatments termined according to Teisseire ability, using the program (2016) also observed that the were composed by the combi- and Guy (2000), with the addi- RStudio 3.2.2. higher temperature, concurrent nation of 4 immersion tempera- tion of 20μl of enzymatic ex- In the biochemical study, with longer imbibition time, tures (25, 50, 75 and 95ºC) × 5 tract, 500μl of potassium phos- the data were subjected to might have caused conforma- sampling times in each associ- phate buffer 50mmol·l-1, pH normality test (Shapiro-Wilk), tional changes in membrane ation (2, 4 , 8, 12 and 24h). 6.5, 250μl pirogalol (1,2,3-ben- with p<0.5. Since the data did phospholipids of of B. brizantha zenotriol) 20mmol·l-1, and hydro- not adjust to any parametrical seeds and, thus, induced solute -1 - Physiological analysis of gen peroxide (H2O2) 5mmol·l , statistic method, graphics lixiviation, about 50,00µS·cm seeds subjected to treatments totaling a volume of 1.0ml. with their standard deviation 1·g-1 higher than the control. to overcome dormancy The formation of purpurogallin were presented, and the aver- In terms of germination per- was measured with a UV- ages compared in a descrip- centage, we observed that there The electrical conductivity of visible spectrophotometer at tive manner. was significant interaction be- the seeds subjected to treat- 430nm. The coefficient of mo- tween the analyzed factors in ments to overcome dormancy lar extinction (2.5mmol·l-1·cm-1) Results and Discussion of P. dubium seeds. Those with was evaluated utilizing four was applied to calculate the no scarification when subjec- replicates of 25 seeds in each specific activity of the enzyme, Physiological analysis of ted to water at 75 and 95ºC treatment. The seeds were which was expressed in mmol seeds treated to overcome showed a significant increase weighted and placed into plastic purpurogallin per min-1·mg-1 of dormancy of this variable, up to 26% cups containing 50ml of deion- protein. higher than at 25ºC (59%). In ized water and kept at 20ºC for The catalase (CAT) enzyme The analysis of the factorial contrast, the seeds with previ- 24h in a biological oxygen de- activity was established accord- design for the variable electri- ous mechanical scarification mand B.O.D. germination ing to Peixoto et al. (1999), cal conductivity (Table I) de- did not show an increase in chamber. After, the electrical with the addition of 20μl of the monstrated there was no sig- germination percentage when conductivity of the water where enzymatic extract, 980μl sodi- nificant interaction between the subjected to the imbibition in the seeds were soaking was um phosphate 0.05mol·l-1 buffer, analyzed factors (with and heated water (Table I). Similar -1 determined using a previously pH 7.0 and H2O2 12.5mmol·l to without previous scarification results where reported for the calibrated conductivity meter, final volume of 1ml. The absor- × temperatures). However, on same species by Dutra et al.

10 JANUARY 2019 • VOL. 44 Nº 1 TABLE I and, on average the seeds took may have led to cell damage, ELECTRICAL CONDUCTIVITY, GERMINATION, MEAN 2.5 days to complete their ger- delaying germination, as ob- GERMINATION TIME (MGT) AND GERMINATION minative process. Yet, for the served in this experiment. SPEED INDEX (GSI), IN SEEDS OF Peltophorum dubium GSI variable there was a sig- With regard to the frequency SUBJECTED TO TREATMENTS WITH AND WITHOUT nificant interaction between the and synchronization it is ob- SCARIFICATION, COMBINED WITH WATER IMMERSION factors; seeds with no scarifi- served that the germination of AT 25, 50, 75 AND 95ºC cation subjected to tempera- P. dubium showed more syn- Non-scarified Scarified tures at 75 and 95ºC showed chrony and uniformity when ºC Average the highest germination speed. the seeds were scarified, pre- Electrical conductivity (µS·cm-1·g-1) According to Carvalho and senting greater initial unimodal 25 121.95 208.44 165.20 b Nakagawa (2012), the higher the peaks, compared to when they 50 134.80 186.42 160.61 b 75 146.79 211.65 179.22 b water temperature used for were not (Figures 1 b, d, f e 95 179.59 228.79 204.19 a overcoming dormancy, the fast- h). Ribeiro et al. (2016) also est the germination will be up observed that the utilization of Average 145.78 B 208.83 A to a certain degree, where the mechanical scarification was C.V. (%) 9.93% temperature instead of increas- enough to achieve germination Germination (%) ing it will impede germination. synchrony, showing unimodal 25 59 b A 69 a A - Alternatively, in the seeds peaks between the second and 50 58 b B 83 a A - treated with scarification and fourth day of the germination. 75 79 a A 80 a A - subjected to heated water, an This demonstrates that in seeds 95 85 a A 70 a A - increase of GSI took place of P. dubium there is a physi- Average - - only at 50ºC, while at higher cal resistance that prevents wa- C.V. (%) 12.43% temperatures (75 and 95ºC) ter intake, and with mechanical MGT (days) germination speed was statisti- scarification of this rigid tegu- 25 2.54 0.92 2.88 a cally similar to the control ment, a more synchronized 50 2.41 2.32 2.36 a (Table I). A hypothesis raised germination is possible. 75 2.10 2.96 2.57 a by Joshi and Chinnusamy 95 2.69 2.57 2.63 a (2014) is that when seeds are Antioxidant enzyme and Average 2.197 A 2.437 A - scarified, fissures in the tegu- lipoperoxide activity in seeds C.V. (%) 16.93% ment are produced, enabling treated to overcome dormancy GSI water inflow to occur faster, and the heated water at high The analysis of specific per- 25 38.58 b A 32.66 b A - temperatures (75 and 95ºC) oxidase activity throughout the 50 36.22 b B 52.57 a A - 75 54.27 a A 38.14 b A - 95 54.97 a A 34.73 b B - Average - - - C.V. (%) 22.28% Average yields followed by the same small letters for different tempera- tures and capital letters for the previous treatments: with and without overcoming dormancy; same letters are not significantly different at the 0.05 level by the Scott-Knot test.

(2012) and Santos et al. (2016), cost and the possibility of being who observed increases of employed at large scale by nurs- 92,2% in emergence and 93,8% erymen, optimizing P. dubium in germination, in seeds soa- seedlings production. ked in heated water at 95ºC. However, Oliveira et al. Examining the water tempera- (2003) and Ribeiro et al. (2016) tures in relation to the scarifica- showed that germination of P. tion of seeds, it can be seen that dubium seeds diminished when the result of hot water at 75 and subjected to water heated at 95ºC is equivalent to the me- 95ºC. The authors considered chanical scarification treatment, that this change in the re- as the seeds that were not scari- sponse to the method for over- fied showed germination per- coming dormancy may occur centages statistically equal to because of the climate condi- those which went through that tions to which the matrix pro- process (Table I). This is a satis- ducer was exposed during seed factory result with regard to maturation, which may alter seedlings production in nursery, the degree of dormancy pre- since overcoming dormancy by sented by the species. Figure 1. Germination frequency and synchronization in seeds of using heated water is an easier In relation to the MGT, there Peltophorum dubium (Sprengel) Tauber, subjected to treatments with and method to use, compared to me- was no significant interaction without scarification, combined with immersion in water at 25, 50, 75 chanical scarification, with low between the analyzed factors and 95ºC.

JANUARY 2019 • VOL. 44 Nº 1 11 process of seed germination of 24h. However, after scarifica- 4, 8, 12 and 24h (Figure 3a). eliminating the free radicals in P. dubium subjected to differ- tion, under the same condi- In contrast, at 75ºC the specif- the P. dubium seeds. ent methods of overcoming tions, the peroxidase activity ic activity of catalase was Under normal conditions, a dormancy is shown in Fi- already increased (1.55umol· about 30 times higher after 8h, balance between the production gure 2. When soaked at 25ºC, min-1·mg-1) within 2h, and and 4 times greater after 24h of ROS and the activity of the the seeds showed a similar during imbibition the activity of imbibition, when compared antioxidant enzymes occurs, behavior with and without pre- declined. In seeds subjected to to the scarified seeds (Fi- especially the peroxidase; yet, vious scarification (Figures 2a, 95ºC without previous scarifi- gure 3e). In the non scarified the increase of ROS causes cell b). A peroxidase activity peak cation (Figures 2g, h), the per- seeds soaked for 12h at 95ºC damage that can lead to cell was observed 4h after imbibi- oxidase had an increment of the rate of the catalase activity death. Therefore, Ribeiro et al. tion, with an increase of 0.54umol·min-1·mg-1 after 4h of was 10 times higher, and at (2014) reported that the in- ~0.20umol·min-1·mg-1 in com- water treatment, keeping similar 24h nearly 24 times higher crease in peroxidase activity parison to 2h. In the seeds im- rates in the following hours. In when compared to the scarified demonstrates an adaptive re- mersed at 50ºC (Figures 2c, d) contrast, when previous scarifi- ones (Figure 3g). sponse to the cell metabolism with no scarification, two ac- cation was done, two peaks of However, at 50ºC higher cat- in regard to the elimination of tivity peaks are observed for that enzyme activity took place, alase rates were found in seeds ROS, allowing the seeds to this enzyme, at 4h and 8h, at 4 and 12h, with raise of subjected to mechanical scari- tolerate stress conditions. For with an increase of ~0.40umol· ~0.50 and 0.80umol·min-1·mg-1, fication, differing from other example, the shock imposed by min-1·mg-1, while in seeds that respectively. treatments, where after 24h of the high water temperatures went through previous scarifi- Analyzing the catalase spe- imbibition, the enzymatic ac- used to overcome seed dor- cation, there is only one activ- cific activity in P. dubium tivity was of 54.97mkat·mg-1, mancy, culminates in higher ity peak of the peroxidase en- seeds (Figures 3a to h), it was while in the seeds without rates of germination, in accor- zyme after 4h of water treat- observed that the seeds that scarification the rate was of dance with the observations in ment, followed by a decrease were not scarified presented 32.06mkat·mg-1 (Figures 3c, d). the P. dubium seeds (Table I). in the following hours. higher rates of this enzyme It was observed that in the The use of high temperatures Immersion of the seeds at activity, when compared to the first hours of water treatment to overcome dormancy of seeds 75ºC with no scarification scarified ones. The seeds soa- (2, 4 and 8h), there was a may initiate a thermal stress in (Figure 2e, f) led to an in- ked at 25ºC without scarifica- higher activity of peroxidase, the cells, both in the reserve crease in peroxidase activity tion, showed a greater catalase while at 12 and 24h the cata- tissues and in the embryo axis, after 4h (1.49umol·min-1·mg-1), specific activity when com- lase had a greater action of increasing the production and which was maintained after pared to the scarified ones at

-1 -1 Figure 2. Specific peroxidase activity (umol·min ·mg ) in seeds of Figure 3. Specific catalase activity (nmol of H2O2 consumed/min/mg protein), Peltophorum dubium, with and without previous scarification, at diffe- in seeds of Peltophorum dubium with and without previous scarification, rent temperatures (25, 50, 75 and 95ºC) after 2, 4, 8, 12 and 24h. at different temperatures (25, 50, 75 and 95ºC) after 2, 4, 8, 12 and 24h.

12 JANUARY 2019 • VOL. 44 Nº 1 the accumulation of ROS, without previous scarification the MDA contents, at 2h peroxidase and catalase specific which in turn will react with (Table I). (18.91nmol·g-1 MDA) and 24h activities, which were able to lipids, proteins and nucleic ac- Prodanović et al. (2007) (20.44nmol·g-1 MDA). revert the lipid peroxidation and ids, resulting in the membrane demonstrated that in seeds of The production of ROS in this way promote germina- lesions caused by lipid peroxi- Picea omorika, which also leads, to lipid peroxidation, tion (79 and 85% respectively). dation. Plants developed a com- have rigid tegument, the in- leading to cell damage. Feng plex system of protection crease of catalase and peroxi- Cai et al. (2011) observed that Conclusion through oxidant detoxification, dase might be involved in the the endosperm of seeds of where the actions of antioxidant protection of the seeds against Jatropha curcas presented an The best methods for over- enzymes (peroxidase, catalase) free radicals, assisting germi- increase in the levels of coming dormancy in P. dubium are essential to neutralize the nation. The authors observed malondialdehyde associated to were the treatments using heat- effects of ROS on the cells ~90% of the germination for the increase of peroxidase af- ed water at 75 and 95ºC in (Joshi and Chinnusamy 2014; this species when the catalase ter 4 and 6 days of germina- non-scarified seeds since, due Harsh et al., 2016). and peroxidase activity dou- tion, followed by catalase to the efficient action of the The H2O2 radical can act as bled, helping in the protection peaks after 6, 8 and 10 days. enzymes catalase and peroxi- a cell marker when present in against the formation of ROS The authors also reported that dase, the levels of lipid peroxi- low concentrations in the cells and promoting the germinative the enzymes doubled their dation were controlled, allow- of the seeds. On the other process. activity during these peaks, ing the seeds to present high hand, in high concentrations its In relation to the lipid perox- highlighting an integrative germination rates and germina- effect is harmful to the cells, idation, we observed the highest activity of the enzymes as tion speed index. since the radical can cross rates in non scarified seeds af- lipid peroxidation increases, membranes, mainly through ter 24h at temperatures of 25, mainly in the endosperm, REFERENCES protein channels as aquaporins 50 and 75ºC. On the other hand, leading to the combat of free and cause oxidative damage to when the seeds were previously radicals and consequently to a Abreu DCA, Porto KG, Nogueira the cell (Das and Roychou- scarified the lipid peroxidation decrease in cell damage. AC (2017) Métodos da supera- dhury, 2014). The action of this takes place after 4h of water This work corroborates previ- ção da dormência e substratos radical in low concentrations in treatment; except at 95ºC, ously described data showing a para germinação de sementes de Tachigali vulgaris L.G. Silva & seeds that are in physical dor- where the non-scarified seeds higher lipid peroxidation for the H.C. Lima. Floram 24: 1-10. mancy, as reported by Gill and after 12h of imbibition was ob- seeds soaked for 12h without Tuteja (2010) in seeds of served, the highest lipoperoxide scarification at 75 and 95ºC, Albuquerque QS, Guimarães RM, -1 Almeida IF, Clemente ACS Capsicum spp., can be benefi- contents (22.67 nmol·g MDA), without any increase in the fol- (2007) Métodos para a supera- cial, acting as a cell wall loos- and in scarified seeds and at lowing hours (Figure 4). This is ção da dormência em sementes ening regulator. It has been 95ºC, there were two peaks in related to the enhancement of de sucupira-preta (Bowdichia observed that this radical can virgilioides KUNTH.). Ciênc. oxidize polysaccharide bonds Agrotecnol. 31: 1716-1721. in the cell wall, leading to its Ataide GM, Borges EL, Gonçalves loosening and, thus, water in- JFC, Guimarães VM, Flores AV flow is facilitated, initiating (2016) Alterações fisiológicas the germination process (El- durante a hidratação de semen- Maarouf-Bouteau and Bailly, tes de Dalbergia nigra ((Vell.) Fr. 2008; Barbosa et al., 2014). All. ex Benth.). Ciênc. Florest. 26: 615-625. Gursky and Santiago (2005) and Ribeiro et al. (2016) ob- AOSA (1983) Seed Vigor Testing Handbook. Association of Official served that the tegument of the Seeds Analysts. East Lasing, MI, seeds of Peltophorum dubium USA. 93 pp. is rigid, having a layer of mac- Barbosa C, Lage P, Vilela A, rosclereids in the exotesta, a Mendes-Faia A, Mendes-Ferreira layer of braquiesclereids in the A (2014) Phenotypic and metabo- mesotesta and a layer of osteo- lic traits of commercial Saccha- sclereids in the endotesta, pro- romyces cerevisiae yeasts. AMB viding its hardness, indicative Expr. 4: 39-57. of physical dormancy. Brasil (2009) Regras para Análise The increase of peroxidase de Sementes. Ministério da and catalase activity in over- Agricultura e Reforma Agrária coming dormancy treatments SNDA/DNDV CLAV. Brasília. with heated water at 75 and 164 pp. 95ºC is indicative that these Brasil (2013) Regras para Análise de enzymes acted to keep low lev- Sementes. Ministério da Agricul- els of the H O radical in the tura, Pecuária e Abastecimento 2 2 MAPA/DAS/ CGAL. Brasília. apoplast of the tegument cells, 70 pp. enabling in this way the loosen- ing of their walls and facilitat- Cai FLan-Ju, M An, XL Shun, G Lin, Fang C (2011) Lipid Pero- ing the water inflow and germi- xidation and Antioxidant Res- nation. These treatments led to Figure 4. Specific lipoperoxide activity (nmol·g-1·of malondialdehyde) in ponses during Seed Germi- the higher germination percent- seeds of Peltophorum dubium with and without previous scarification, at nation of Jatropha curcas. Int. ages (79 and 85%, respectively) different temperatures (25, 50, 75 and 95ºC) after 2,4,8,12 and 24h. J. Agric. Biol. 13: 25-30.

JANUARY 2019 • VOL. 44 Nº 1 13 Cardoso ED, Sá ME, Haga KI, de (Peltophorum dubium (Spreng.) peroxidation and activities of pe- (Sprengel) Taubert subm etidas Binotti FFS, Nogueira DC, Taub). In 56º Congr. Nac. de Bo- roxidase and polyphenoloxidase a tratamentos de superação Filho WVV (2014) Desempenho tânica. Sociedade Botânica do in seeds of Telfairia occidentalis. de dormência. J. Agron. Sci. fisiológico e superação de dor- Brasil. Resumos, Vol. 1. Curitiba, Seed Sci. Technol. 28: 1-9. 5: 158-169. mência em sementes de Bra- Brazil. Perez SCJGA, Negreiros GF (2001) Ribeiro R.C, Matias JR, Pelacani chiaria brizantha submetidas a Guglieri-Caporal A, Caporal FJM, Efeitos do pré-condicionamento na CR, Dantas BF (2014) Activity tratamento químico e envelheci- Pott A, Vinci-Carlos HC, viabilidade e no vigor de sementes of antioxidant enzymes and mento artificial. Semina: Cs. Morales CAS (2011). 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