- Email: [email protected]
Comparative assessment for the effects of reactive species on seed germination, growth and metabolisms of vegetables
Scientia Horticulturae 227 (2018) 85–91
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Research Paper
Comparative assessment for the effects of reactive species on seed germination, growth and metabolisms of vegetables
MARK
⁎
Kamonporn Panngoma, , Thanyarat Chuesaarda, Natchathorn Tamchanb, Thitima Jiwchanb, ⁎ Kantirat Srikongsritongc, Gyungsoon Parkd, a
Basic Science, Maejo University Phrae Campus, Rong Kwang, Phrae, 54140, Thailand Agroforestry, Maejo University Phrae Campus, Rong Kwang, Phrae, 54140, Thailand c Plant Production Technology, Maejo University Phrae Campus, Rong Kwang, Phrae, 54140, Thailand d Plasma Bioscience Research Center, Kwangwoon University, Seoul, 01897, Korea b
A R T I C L E I N F O
A B S T R A C T
Keywords: Reactive oxygen and nitrogen species Seed germination Seedling growth Seed metabolism Vegetables
Reactive oxygen and nitrogen species (RONS) play an important role as signaling molecules in various biological pathways in plants. The aim of this study is to assess the effects of H2O2 (ROS) and nitric oxide (RNS) on seed germination, seedling growth and metabolism of two vegetables, coriander and carrot. Seed germination and seedling length of vegetables were significantly increased after treatment with H2O2 for 12 and 24 h. An optimal concentration of H2O2 giving maximum effect was 25–50 mM for both vegetables. The seed germination and seedling growth of coriander were significantly increased after treatment with 12.5 and 25 μM sodium nitroprusside (SNP; generating about 10–25 μM NO) for 12 and 24 h. On the other hand, SNP treated carrot seeds showed no significant difference in seed germination, compared to control. Amounts of total soluble proteins extracted from both vegetables at 72 h post-germination were significantly greater in treatment with 25 mM H2O2. Amounts of total soluble proteins were significantly increased in coriander seeds treated with SNP during germination but not in carrot. The α-amylase activity measured as the amount of reducing sugar was significantly increased in coriander and carrot after H2O2 treatment. Our results suggest that the effect of RONS on seed germination, growth and development, and seed metabolisms can be various depending on dose and plant species.
1. Introduction Vegetables are an important source for healthy foods because of their richness in nutrients and vitamins. Coriander (Coriandum sativum L.), a member belonging to the Apiaceae family, is an annual herbaceous vegetable and usually used as a flavoring worldwide ingredient in several foods (Laribi et al., 2015). Coriander has been also used in traditional medicinal treatment such as aromatherapy and drugs (Msaada et al., 2017) because they contain a lot of essential oils and phenolic compounds, especially in fruit part (Laribi et al., 2015; Msaada et al., 2017). Carrot (Daucus carota L.), a well-known root vegetable and also a member of Apiaceae family, contains many antioxidant compounds such as numerous α and β carotenes and provitamin A (Silva Dias, 2014). In spite of being useful sources for nutrition and medicine, coriander and carrot often exhibit poor efficiency in production yield because of the low and inconsistent rate of seed germination (Rithichai et al., 2009; Pereira et al., 2008). Low quality and vigor of coriander seeds may be caused by the staggered flowering behavior of plant ⁎
(Rithichai et al., 2009). Quality of carrot seeds is highly variable depending on umbel order of inflorescence and this may cause inconsistent germination efficiency (Pereira et al., 2008). Moreover, coriander and carrot seeds require highly optimized environmental conditions for efficient germination such as moisture, temperature, light, pH and oxygen (Koger et al., 2004; Pereira et al., 2008). Seed germination as an initial step of growth and development in plants is the most important stage. During seed germination process, stored nutrients inside an endosperm (monocot plants) or cotyledon (dicot plants) are catabolized by degradation enzymes to produce energy (Gallardo et al., 2001). On the other hand, the anabolic pathway such as protein synthesis is progressed inside the seeds after imbibition (Miransari and Smith, 2014). These metabolic pathways are triggered by water uptake of seeds as well as oxygen gas diffused into the seed (Gallardo et al., 2001). Some plant seeds go through a long term dormancy after ripening and are very hard to germinate because of thicker and stronger seed coat, germination inhibitors on seed surface, hormone level and unsuitable environmental conditions (Arc et al., 2013).
Corresponding authors. E-mail addresses: [email protected], [email protected] (K. Panngom), [email protected] (G. Park).
http://dx.doi.org/10.1016/j.scienta.2017.09.026 Received 16 June 2017; Received in revised form 28 August 2017; Accepted 24 September 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
Scientia Horticulturae 227 (2018) 85–91
K. Panngom et al.
The structure of seed coat is important for water absorption and oxygen gas penetration during germination stage (Gutterman, 1994). Indeed, phytohormones, especially gibberellins (GA) and abscisic acid (ABA), are key players for controlling seed germination (gibberellins) and dormancy (abscisic acid) (Bewley, 1997). Several chemical methods have been tried for breaking seed dormancy to enhance seed germination rate, for example, soaking seeds in saline solutions (Panuccio et al., 2014), and treating seeds with plasma generated reactive species (Ji et al., 2015) and chemically produced reactive species (Barba-Espin et al., 2012). Reactive species in plants can play dual roles, beneficial or harmful, depending on the amount. They are essential for stimulating physiological and developmental processes and resisting stresses (Liu et al., 2007), whereas high level of reactive species triggers senescence, cell death, cell cycle arrest and other harmful reactions (Graves, 2012). Chemically generated and exogenously applied reactive species can play critical roles during plant development such as seed germination and growth as demonstrated in various plants; rice, barley, legume, and maize (Liu et al., 2007; Gondim et al., 2010; Cavusoglu and Kabar, 2010; Barba-Espin et al., 2012). These studies suggest that exogenous reactive species enable to induce and stimulate seed germination and subsequent seedling growth. However, there are still limited number of studies on comparative analysis among individual reactive species in determining effectiveness as an activator and interaction with plant species. In this study, we compared the effectiveness of chemically generated RONS in enhancing seed germination, seedling growth and seed metabolisms of two vegetable plants in the Apiaceae family, coriander and carrot. The results from this study will provide the useful information in understanding the effectiveness of individual reactive species on plant development and the interaction between reactive species and plant species.
The percentage of seed germination =
Number of seeds germinated x 100 Total number of seed
In addition, germination index (GI) was also calculated for assessing seed vigor as described in below;
Germination index =
No. of seed germination Day of first count No. of seed germination + …+ Day of final count
In the case of seedling growth, we randomly selected 10 coriander (6 days old) and 10 carrot (5 days old) seedling plants in each treatment, and then measured the seedling length. 2.4. Analysis for the amount of total soluble proteins and reducing sugars Coriander and carrot seeds were treated with H2O2 and SNP solutions as described in earlier section. Geminated seeds were harvested 0 and 72 h after H2O2 and SNP treatment. Then, seeds were dried by incubating at 60 °C for 24 h and then grinded in liquid nitrogen. To extract total soluble proteins, 6 ml of Tris-buffer (0.05 M Tris hydroxylmethionine, 0.02 M CaCl2, adjust pH 7.4 by using 6 M HCl) solution was added to grinded powder and the mixture was shaken on platform mixer for 1 h. Homogenates were centrifuged at 2879 × g for 5 min. Supernatant was transferred to a new tube and kept on ice. Concentration of proteins was measured at 280 nm using spectrophotometer. Reducing sugar amounts were measured by dinitro-salicylic (DNS) method. Extracted solutions (0.25 ml) were mixed with Tris-buffer (0.25 ml), and then 0.5 ml starch solution (1% w/v) (starch prepared in 0.02 M phosphate buffer pH 7.0) was added. Mixture was incubated at 37 °C for 1 h. Then, 0.5 ml of DNS (1% (w/v) 3,5-dinitrosalic acid, 0.02 M NaOH, 30% (w/v) sodium potassium tartrate solution was added to the mixture for detection of reducing sugars and incubated at 100 °C for 5 min. After 5 ml of distilled water was added to samples, absorbance was measured at 540 nm using spectrophotometer. The amounts of reducing sugar were calculated from a standard curve made by using maltose as a reference sugar.
2. Materials and methods 2.1. Plant materials and growth condition Coriander and carrot seeds were used for testing the effect of chemically generated reactive species on seed germination, growth and metabolisms. Variety 3A (AAA) and 333 seeds were used for coriander and carrot, respectively. Germinated seeds were planted in sterilized soil and kept in the natural greenhouse of Agroforestry program, Maejo University Phrae Campus, Thailand.
2.5. Statistical analysis The data of seed germination rate, seedling length, and concentration of total soluble proteins and reducing sugars were expressed as mean and standard error (SE) of the mean for indicated number of replicates (≥3). Statistical analysis was performed by using student’s ttest to establish significance between data points, and significant differences were based on the p < 0.05 or p < 0.01 (*p < 0.05 and **p < 0.01).
2.2. Treatment with chemically generated reactive species All chemicals used in the experiments were analytical grade. H2O2 solution and sodium nitroprusside (SNP) were used as a donor for H2O2 and NO, respectively. For H2O2 treatment on coriander and carrot seeds, seeds (150 seeds per treatment) were placed in a beaker containing 30 ml of H2O2 solution of 25, 50, 100, and 200 mM and incubated at room temperature for 12 and 24 h. For NO treatment, we dissolved sodium nitroprusside (SNP) in distilled water to make the concentration of 12.5, 25, 50 and 100 μM. After placing coriander and carrot seeds in the beakers containing SNP solutions, the beakers were incubated for 12 and 24 h. After incubation, seeds were moved onto two layers of wet-cultivate paper in petri-dish (∅ 9 centimeters) and then germination was monitored every day.
3. Results 3.1. Exogenous H2O2 enhanced seed germination and seedling growth of both vegetables Coriander and carrot seeds were treated with several concentrations of H2O2 solution under room temperature. The pH of all H2O2 solutions before and after soaking seeds was between 5.43 and 5.59. Fig. 1A shows the percentage of coriander seed germination after treatment with H2O2 for 12 and 24 h. The percentage of seed germination was significantly increased after treatment with all concentrations of H2O2 solutions. Particularly, 25 mM H2O2 solution showed the greatest effect; 82 and 87% germination after 12 and 24 h, respectively. In the treatment with 200 mM H2O2 solution, the percentage of seed germination was initially reduced but became higher than that of control (no H2O2) after 120 h. Fig. 1B shows the morphology of germinated coriander seeds. Radicle of embryo was emerged through seed coat earlier in seeds treated for 12 h than 24 h, Germination index was also
2.3. Seed germination and seedling growth analysis For analysis of coriander and carrot seed germination, we counted number of germinated seeds every 24 h. Germinated seed was determined by the emergence of the radicle from seed coat. Seed germination rate was calculated as follows; 86
Scientia Horticulturae 227 (2018) 85–91
K. Panngom et al.
Fig. 1. The effect of H2O2 on seed germination of coriander. (A) The percentage of seed germination after treatment with H2O2; 25, 50, 100 and 200 mM for 12 and 24 h. (B) The morphology of germinated coriander seeds after 48 and 96 h. (C) Germination index (GI) of coriander seeds. *p < 0.05 or **p < 0.01.
(Fig. 2A). Differently from coriander seeds, carrot seeds germinated faster in the treatment for 24 h than 12 h as shown in Fig. 2A and B. Radicles of carrot seeds treated for 24 h were emerged from seed coat earlier and longer, compared to the 12 h treated seeds (Fig. 2B). Moreover, germination index of carrot seeds was significantly increased after treatment with 25 and 50 mM H2O2 for 24 h (Fig. 2C). These results indicate that carrot seeds require higher concentration and longer treatment of H2O2 than coriander seeds.
significantly increased in coriander seeds treated with H2O2 solutions with the highest effect in 25–50 mM (Fig. 1C). For carrot seeds treated with H2O2 solutions, the percentage of seed germination was significantly increased and 25 and 50 mM concentrations resulted in the highest effect in both 12 and 24 h treatment (Fig. 2A). The percentage of germination was up to 71% in the treatment for 24 h (Fig. 2A). Increase in germination percentage was slowed down when the concentration of H2O2 was higher than 50 mM
Fig. 2. The effect of H2O2 on seed germination of carrot. (A) The percentage of seed germination after treatment with H2O2; 25, 50, 100 and 200 mM for 12 and 24 h. (B) The morphology of germinated carrot seeds after 24 and 48 h. (C) Germination index (GI) of carrot seeds. *p < 0.05 or **p < 0.01.
87
Scientia Horticulturae 227 (2018) 85–91
K. Panngom et al.
Seedling growth was analyzed by measuring the length of seedlings after 5 (carrot) and 6 (coriander) days. The seedling length of coriander was significantly increased after H2O2 treatment for both 12 and 24 h although the increase was slower in higher concentration of H2O2 and 24 h treatment (Fig. 3A). For carrot plant, the seedling length was also increased significantly after H2O2 treatment and there was no significant difference among H2O2 concentration and treatment time (Fig. 3B). 3.2. Exogenous NO enhances seed germination and seedling growth of coriander more greatly than carrot In order to compare the effects between ROS and RNS on seed germination and seedling growth, we treated coriander and carrot seeds with nitric oxide (NO) generated from SNP (sodium nitroprusside). The pH of SNP solutions before and after soaking seeds was between 5.18 and 5.35. Germination percentage of coriander seeds was significantly increased after SNP treatment and the increase was faster in 12.5 μM treatment for both 12 (79%) and 24 (84.5%) hrs (Fig. 4A). The radicle emergence from seed coat was slightly earlier in 12 h treatment (Fig. 4B). Germination index of coriander seeds treated with SNP was significantly higher than control in both 12 and 24 h treatment (Fig. 4C). Carrot seeds treated with SNP exhibited no dramatic changes in germination percentage compared to control although increased germination was observed in the treatments with several concentrations of SNP (Fig. 5A and B). Generally, germination level was slightly lower in 24 than 12 h treatment (Fig. 5A). Germination index of SNP treated seeds was not significantly different from that of control in both 12 and 24 h treatment (Fig. 5C). These results demonstrate that carrot seeds seem to respond more sensitively to H2O2 than NO generated from SNP in terms of seed germination. To determine the effect of RNS on plant growth, we assessed the influence of SNP treatment on seedling growth by measuring the length. The length of coriander seedlings germinated from seeds treated with SNP was significantly increased with the greater effect in 12.5 and 25 μM treatment for 12 h (Fig. 6A). In case of carrot, seedlings length was longer in SNP treatment for 12 h than in control even though seed germination was not significantly affected by SNP treatment (Fig. 6B). Longer radicles were observed in both coriander and carrot plants in the treatments with 12.5 and 25 μM SNP for 12 h (Fig. 6A and B). In addition, seedling growth was more dramatically increased in coriander than carrot in response to SNP treatment. 3.3. H2O2 and NO treatment can modulate seed metabolisms during germination To elucidate the distinctive effect of RONS on seed metabolisms during germination, we measured the level of total soluble proteins and reducing sugar (α-amylase activity indicator) in germinating seeds at 0 and 72 h after treatment with H2O2 and SNP for 24 h. Amounts of total soluble proteins in coriander and carrot seeds after H2O2 treatment were significantly increased (Fig. 7A). After 72 h, protein level in coriander seeds was slightly reduced except 25 mM treatment (Fig. 7A). However, carrot showed the increased level of soluble proteins even after 72 h (Fig. 7A). In SNP treatment, concentration of total soluble proteins was significantly increased in coriander, especially in 12.5 and 25 μM SNP treatment (Fig. 7B). In carrot, no significant change in protein level was observed in all treatments (Fig. 7B). The quantity of reducing sugar in coriander seeds was significantly increased at 72 h post germination in treatments with 50 and 100 mM H2O2 solutions (Fig. 8A). In carrot, higher level of reducing sugar was observed in treatments with most H2O2 concentrations and in both 0 and 72 h (Fig. 8A). However, treatment with NO generated from SNP did not cause any change in the level of reducing sugar in both coriander and carrot (Fig. 8B). These results suggest that seed metabolisms
Fig. 3. The length of coriander and carrot seedlings grown from seeds treated with H2O2 for 6 days. (A) The length and morphology of coriander seedlings. (B) The length and morphology of carrot seedlings. *p < 0.05 or **p < 0.01.
88
Scientia Horticulturae 227 (2018) 85–91
K. Panngom et al.
Fig. 4. The effect of NO generated from SNP on germination of coriander seeds. (A) The percentage of seed germination after treatment with NO generated from SNP in concentrations; 12.5, 25, 50 and 100 μM for 12 and 24 h. (B) The morphology of germinated coriander seeds after 48 and 96 h. (C) Germination index (GI) of coriander seeds. *p < 0.05 or **p < 0.01.
plant species. In our results, exogenously applied H2O2 solutions can increase the efficiency of seed germination and subsequent seedling development in both coriander and carrot. However, NO generated from SNP showed greater activation effect on coriander than carrot. Carrot exhibited the reduced level of H2O2 effect on germination and growth compared to coriander. This may be because carrot has higher scavenging or antioxidant activity for H2O2 and NO than coriander.
can be differently affected by exogenous RONS depending on plant species and individual reactive species. 4. Discussion Although RONS are known as signaling molecules in various cellular processes, our results suggest that RONS can act differently in different
Fig. 5. The effect of NO generated from SNP on germination of carrot seeds. (A) The percentage of seed germination after treatment with NO generated from SNP in concentrations; 12.5, 25, 50 and 100 μM for 12 and 24 h. (B) The morphology of germinated carrot seeds after 24 and 48 h. (C) Germination index (GI) of carrot seeds. *p < 0.05 or **p < 0.01.
89
Scientia Horticulturae 227 (2018) 85–91
K. Panngom et al.
Fig. 7. The amount of total soluble proteins in germinated coriander and carrot seeds treated with H2O2 and NO generated from SNP (24 h) after 0 and 72 h post-germination. *p < 0.05 or **p < 0.01.
Thus, treatment with more H2O2 and NO may be required to achieve similar level of developmental activation in carrot. Possible functions of H2O2 and NO during germination have been suggested (Barba-Espin et al., 2010; Barba-Espin et al., 2012; Liu et al., 2010; Kopyra and Gwozdz, 2003). H2O2 is known to induce ABA catabolism and GA synthesis and this may stimulate early seed germination in plants (Barba-Espin et al., 2010; Barba-Espin et al., 2012; Liu et al., 2010). NO stimulates the activity of α-amylase during germination (Kopyra and Gwozdz, 2003). In our study, the level of reducing sugar, an indicator for α-amylase activity, in germinating seeds was not affected by NO treatment although H2O2 treatment promoted the generation of reducing sugar in both coriander and carrot. H2O2 treatment may enhance the activity of α-amylase probably through GA hormone induction in both coriander and carrot. However, increase in the level of reducing sugar was more dramatic in carrot than coriander although germination was more greatly enhanced by H2O2 in coriander. This may be because data of reducing sugar amount is not enough to assess the activity of α-amylase or another mechanism(s) is possibly involved in H2O2 action. About action mechanism(s) of NO during seed germination, it seems to be various depending on plant species. In our study, carrot is not affected by NO treatment. In addition, the amount of reducing sugar was not changed after NO treatment in both coriander and carrot. Since NO is a well-known regulator for abiotic and biotic stresses, the influence of NO on plant development may demonstrate broad spectrum of outcomes in various plant species. From our study, it was not clearly known how NO could stimulate germination of coriander seeds. Besides activation of α-amylase, another mechanism(s)
Fig. 6. The length of coriander and carrot seedlings grown from seeds treated with NO generated from SNP for 6 days. (A) The length and morphology of coriander seedlings. (B) The length and morphology of carrot seedlings. *p < 0.05 or **p < 0.01.
90
Scientia Horticulturae 227 (2018) 85–91
K. Panngom et al.
and seedling growth in carrot while NO generated from SNP did not cause any significant change. Differential effects on plant development were generated depending on plant species and dose of individual reactive species. Our results provide useful information in understanding comparative influence of two reactive species on early development of vegetable species. Conflict of interest The authors declare no conflict of interest. Acknowledgment This work was partially supported by the grants project from Maejo University Phrae Campus in the year of 2015 (MJ.2-58-005). References Arc, E., Galland, M., Godin, B., Cueff, G., Rajjou, L., 2013. Nitric oxide implication in the control of seed dormancy and germination. Front. Plant Sci. 4, 346. Barba-Espin, G., Diaz-Vivancos, P., Clemente-Moreno, M.J., Albacete, A., Faize, L., Faize, M., Perez-Alfocea, F., Hernandez, J.A., 2010. Interaction between hydrogen peroxide and plant hormones during germination and the early growth of pea seedlings. Plant Cell Environ. 33, 981–994. Barba-Espin, G., Hernandez, J.A., Diaz-Vivancos, P., 2012. Role of H2O2 in pea seed germination. Plant Signal. Behav. 7 (2), 193–195. Bewley, J.D., 1997. Seed germination and dormancy. The Plant Cell 9, 1055–1066. Cavusoglu, K., Kabar, K., 2010. Effects of hydrogen peroxide on the germination and early seedling growth of barley under NaCl and high temperature stresses. Eurasia J. Biosci. 4, 70–79. Gallardo, K., Job, C., Groot, S.P.C., Puype, M., Demol, H., Vandekerckhove, J., Job, D., 2001. Proteomic analysis of Arabidopsis seed germination and priming. Plant Physiol. 126, 835–884. Gondim, F.A., Gomes-Filho, E., Lacerda, C.F., Prisco, J.T., Azevedo Neto, A.D., Marques, E.C., 2010. Pretreatment with H2O2 in maize seeds: effects on germination and seedling acclimation to salt stress. Braz. J. Plant Physiol. 22 (2), 103–112. Graves, D.B., 2012. The emerging role of reactive oxygen and nitrogen species in redox biology and some implications for plasma applications to medicine and biology. Appl. Phys. 45, 263001. Gutterman, Y., 1994. Strategies of seed dispersal and germination in plants inhabiting deserts. Bot. Rev. 60, 373. Ji, S.H., Kim, T., Panngom, K., Hong, Y.J., Pengkit, A., Park, D.H., Kang, M.H., Lee, S.H., Im, J.S., Kim, J.S., Uhm, H.S., Choi, E.H., Park, G., 2015. Assessment of the effects of nitrogen plasma and plasma-generated nitric oxide on early development of Coriandum sativum. Plasma Process. Polym. 12, 1164–1173. Koger, C.H., Reddy, K.N., Poston, D.H., 2004. Factors affecting seed germination, seedling emergence and survival of Texas weed (Caperonia palustris). Weed Sci. 52, 989–995. Kopyra, M., Gwozdz, E.A., 2003. Nitric oxide stimulates seed germination and counteracts the inhibitory effect of heavy metals and salinity on root growth of Lupinus luteus. Plant Physiol. Biochem. 41, 1011–1017. Laribi, B., Kouki, K., Hamdi, M., Bettaieb, T., 2015. Coriander (Coriandrum sativum L.) and its bioactive constituents. Fitoterapia 103, 9–26. Liu, H.Y., Yu, X., Cui, D.Y., Sun, M.H., Sun, W.N., Tang, Z.C., Kwak, S.S., Su, W.A., 2007. The role of water channel proteins and nitric oxide signaling in rice seed germination. Cell Res. 17, 638–649. Liu, Y., Ye, N., Liu, R., Chen, M., Zhang, J., 2010. H2O2 mediates the regulation of ABA catabolism and GA biosynthesis in Arabidopsis seed dormancy and germination. J. Exp. Bot. 61 (11), 2979–2990. Miransari, M., Smith, D.L., 2014. Plant hormones and seed germination. Environ. Exp. Bot. 99, 110–121. Msaada, K., Jemia, M.B., Salem, N., Bachrouch, O., Sriti, J., Tammar, S., Bettaieb, I., Jabri, I., Kefi, S., Limam, F., Marzouk, B., 2017. Antioxidant activity of methanolic extracts from three coriander (Coriandrum sativum L.) fruit varieties. Arab. J. Chem. 10 (2), S3176–S3183. Panuccio, M.R., Jacobsen, S.E., Akhtar, S.S., Muscolo, A., 2014. Effect of saline water on seed germination and early seedling growth of the halophyte quinoa. AoB PLANTS 6, plu047. Pereira, R.S., Nascimento, W.M., Vieira, J.V., 2008. Carrot seed germination and vigor in response to temperature and umbel orders. Sci. Agric. 65 (2), 145–150. Rithichai, P., Sampantharat, P., Jirakiattikul, Y., 2009. Coriander (Coriandrum sativum L.) seed quality as affected by accelerated aging and subsequent hydropriming. Asian J. Food Agro-Ind. (Special Issue), S217–S221. Silva Dias, J.C., 2014. Guiding strategies for breeding vegetable cultivars. Agric. Sci. 5, 9–32. Weitbrecht, K., Müller, K., Leubner-Metzger, G., 2011. First off the mark: early seed germination. J. Exp. Bot. 62 (10), 3289–3309.
Fig. 8. The amounts of reducing sugar in germinated coriander and carrot seeds treated with H2O2 and NO generated from SNP (24 h) after 0 and 72 h post-germination. *p < 0.05 or **p < 0.01.
may be possibly involved, but further intense investigation will be needed. Our results demonstrate that H2O2 and NO can activate biochemical changes in seeds during germination. Biochemical changes during seed germination are well demonstrated in studies; during seed imbibition, metabolisms of stored proteins (especially enzymes) can be activated in seeds and also energy-related enzymes for major metabolic pathways play important roles in inducing early seed germination in plant (Weitbrecht et al., 2011). The level of total soluble proteins in germinating carrot and coriander seeds is significantly increased after H2O2 treatment. NO treatment elevates the amount of total soluble proteins in coriander but not in carrot. These results suggest that gene expression inside coriander and carrot seeds is stimulated by exogenous H2O2 and NO even though carrot responds to NO less sensitively. It may be possible that NO level used in the treatment is not enough to induce molecular and developmental activation in carrot. 5. Conclusion Effects of exogenous H2O2 and NO generated from SNP on seed germination, growth and development of seedlings, and seed metabolisms during germination were analyzed in coriander and carrot. Both H2O2 and NO induced enhancement in germination and seedling growth in coriander. However, only H2O2 promoted seed germination
91
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Research Paper
Comparative assessment for the effects of reactive species on seed germination, growth and metabolisms of vegetables
MARK
⁎
Kamonporn Panngoma, , Thanyarat Chuesaarda, Natchathorn Tamchanb, Thitima Jiwchanb, ⁎ Kantirat Srikongsritongc, Gyungsoon Parkd, a
Basic Science, Maejo University Phrae Campus, Rong Kwang, Phrae, 54140, Thailand Agroforestry, Maejo University Phrae Campus, Rong Kwang, Phrae, 54140, Thailand c Plant Production Technology, Maejo University Phrae Campus, Rong Kwang, Phrae, 54140, Thailand d Plasma Bioscience Research Center, Kwangwoon University, Seoul, 01897, Korea b
A R T I C L E I N F O
A B S T R A C T
Keywords: Reactive oxygen and nitrogen species Seed germination Seedling growth Seed metabolism Vegetables
Reactive oxygen and nitrogen species (RONS) play an important role as signaling molecules in various biological pathways in plants. The aim of this study is to assess the effects of H2O2 (ROS) and nitric oxide (RNS) on seed germination, seedling growth and metabolism of two vegetables, coriander and carrot. Seed germination and seedling length of vegetables were significantly increased after treatment with H2O2 for 12 and 24 h. An optimal concentration of H2O2 giving maximum effect was 25–50 mM for both vegetables. The seed germination and seedling growth of coriander were significantly increased after treatment with 12.5 and 25 μM sodium nitroprusside (SNP; generating about 10–25 μM NO) for 12 and 24 h. On the other hand, SNP treated carrot seeds showed no significant difference in seed germination, compared to control. Amounts of total soluble proteins extracted from both vegetables at 72 h post-germination were significantly greater in treatment with 25 mM H2O2. Amounts of total soluble proteins were significantly increased in coriander seeds treated with SNP during germination but not in carrot. The α-amylase activity measured as the amount of reducing sugar was significantly increased in coriander and carrot after H2O2 treatment. Our results suggest that the effect of RONS on seed germination, growth and development, and seed metabolisms can be various depending on dose and plant species.
1. Introduction Vegetables are an important source for healthy foods because of their richness in nutrients and vitamins. Coriander (Coriandum sativum L.), a member belonging to the Apiaceae family, is an annual herbaceous vegetable and usually used as a flavoring worldwide ingredient in several foods (Laribi et al., 2015). Coriander has been also used in traditional medicinal treatment such as aromatherapy and drugs (Msaada et al., 2017) because they contain a lot of essential oils and phenolic compounds, especially in fruit part (Laribi et al., 2015; Msaada et al., 2017). Carrot (Daucus carota L.), a well-known root vegetable and also a member of Apiaceae family, contains many antioxidant compounds such as numerous α and β carotenes and provitamin A (Silva Dias, 2014). In spite of being useful sources for nutrition and medicine, coriander and carrot often exhibit poor efficiency in production yield because of the low and inconsistent rate of seed germination (Rithichai et al., 2009; Pereira et al., 2008). Low quality and vigor of coriander seeds may be caused by the staggered flowering behavior of plant ⁎
(Rithichai et al., 2009). Quality of carrot seeds is highly variable depending on umbel order of inflorescence and this may cause inconsistent germination efficiency (Pereira et al., 2008). Moreover, coriander and carrot seeds require highly optimized environmental conditions for efficient germination such as moisture, temperature, light, pH and oxygen (Koger et al., 2004; Pereira et al., 2008). Seed germination as an initial step of growth and development in plants is the most important stage. During seed germination process, stored nutrients inside an endosperm (monocot plants) or cotyledon (dicot plants) are catabolized by degradation enzymes to produce energy (Gallardo et al., 2001). On the other hand, the anabolic pathway such as protein synthesis is progressed inside the seeds after imbibition (Miransari and Smith, 2014). These metabolic pathways are triggered by water uptake of seeds as well as oxygen gas diffused into the seed (Gallardo et al., 2001). Some plant seeds go through a long term dormancy after ripening and are very hard to germinate because of thicker and stronger seed coat, germination inhibitors on seed surface, hormone level and unsuitable environmental conditions (Arc et al., 2013).
Corresponding authors. E-mail addresses: [email protected], [email protected] (K. Panngom), [email protected] (G. Park).
http://dx.doi.org/10.1016/j.scienta.2017.09.026 Received 16 June 2017; Received in revised form 28 August 2017; Accepted 24 September 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
Scientia Horticulturae 227 (2018) 85–91
K. Panngom et al.
The structure of seed coat is important for water absorption and oxygen gas penetration during germination stage (Gutterman, 1994). Indeed, phytohormones, especially gibberellins (GA) and abscisic acid (ABA), are key players for controlling seed germination (gibberellins) and dormancy (abscisic acid) (Bewley, 1997). Several chemical methods have been tried for breaking seed dormancy to enhance seed germination rate, for example, soaking seeds in saline solutions (Panuccio et al., 2014), and treating seeds with plasma generated reactive species (Ji et al., 2015) and chemically produced reactive species (Barba-Espin et al., 2012). Reactive species in plants can play dual roles, beneficial or harmful, depending on the amount. They are essential for stimulating physiological and developmental processes and resisting stresses (Liu et al., 2007), whereas high level of reactive species triggers senescence, cell death, cell cycle arrest and other harmful reactions (Graves, 2012). Chemically generated and exogenously applied reactive species can play critical roles during plant development such as seed germination and growth as demonstrated in various plants; rice, barley, legume, and maize (Liu et al., 2007; Gondim et al., 2010; Cavusoglu and Kabar, 2010; Barba-Espin et al., 2012). These studies suggest that exogenous reactive species enable to induce and stimulate seed germination and subsequent seedling growth. However, there are still limited number of studies on comparative analysis among individual reactive species in determining effectiveness as an activator and interaction with plant species. In this study, we compared the effectiveness of chemically generated RONS in enhancing seed germination, seedling growth and seed metabolisms of two vegetable plants in the Apiaceae family, coriander and carrot. The results from this study will provide the useful information in understanding the effectiveness of individual reactive species on plant development and the interaction between reactive species and plant species.
The percentage of seed germination =
Number of seeds germinated x 100 Total number of seed
In addition, germination index (GI) was also calculated for assessing seed vigor as described in below;
Germination index =
No. of seed germination Day of first count No. of seed germination + …+ Day of final count
In the case of seedling growth, we randomly selected 10 coriander (6 days old) and 10 carrot (5 days old) seedling plants in each treatment, and then measured the seedling length. 2.4. Analysis for the amount of total soluble proteins and reducing sugars Coriander and carrot seeds were treated with H2O2 and SNP solutions as described in earlier section. Geminated seeds were harvested 0 and 72 h after H2O2 and SNP treatment. Then, seeds were dried by incubating at 60 °C for 24 h and then grinded in liquid nitrogen. To extract total soluble proteins, 6 ml of Tris-buffer (0.05 M Tris hydroxylmethionine, 0.02 M CaCl2, adjust pH 7.4 by using 6 M HCl) solution was added to grinded powder and the mixture was shaken on platform mixer for 1 h. Homogenates were centrifuged at 2879 × g for 5 min. Supernatant was transferred to a new tube and kept on ice. Concentration of proteins was measured at 280 nm using spectrophotometer. Reducing sugar amounts were measured by dinitro-salicylic (DNS) method. Extracted solutions (0.25 ml) were mixed with Tris-buffer (0.25 ml), and then 0.5 ml starch solution (1% w/v) (starch prepared in 0.02 M phosphate buffer pH 7.0) was added. Mixture was incubated at 37 °C for 1 h. Then, 0.5 ml of DNS (1% (w/v) 3,5-dinitrosalic acid, 0.02 M NaOH, 30% (w/v) sodium potassium tartrate solution was added to the mixture for detection of reducing sugars and incubated at 100 °C for 5 min. After 5 ml of distilled water was added to samples, absorbance was measured at 540 nm using spectrophotometer. The amounts of reducing sugar were calculated from a standard curve made by using maltose as a reference sugar.
2. Materials and methods 2.1. Plant materials and growth condition Coriander and carrot seeds were used for testing the effect of chemically generated reactive species on seed germination, growth and metabolisms. Variety 3A (AAA) and 333 seeds were used for coriander and carrot, respectively. Germinated seeds were planted in sterilized soil and kept in the natural greenhouse of Agroforestry program, Maejo University Phrae Campus, Thailand.
2.5. Statistical analysis The data of seed germination rate, seedling length, and concentration of total soluble proteins and reducing sugars were expressed as mean and standard error (SE) of the mean for indicated number of replicates (≥3). Statistical analysis was performed by using student’s ttest to establish significance between data points, and significant differences were based on the p < 0.05 or p < 0.01 (*p < 0.05 and **p < 0.01).
2.2. Treatment with chemically generated reactive species All chemicals used in the experiments were analytical grade. H2O2 solution and sodium nitroprusside (SNP) were used as a donor for H2O2 and NO, respectively. For H2O2 treatment on coriander and carrot seeds, seeds (150 seeds per treatment) were placed in a beaker containing 30 ml of H2O2 solution of 25, 50, 100, and 200 mM and incubated at room temperature for 12 and 24 h. For NO treatment, we dissolved sodium nitroprusside (SNP) in distilled water to make the concentration of 12.5, 25, 50 and 100 μM. After placing coriander and carrot seeds in the beakers containing SNP solutions, the beakers were incubated for 12 and 24 h. After incubation, seeds were moved onto two layers of wet-cultivate paper in petri-dish (∅ 9 centimeters) and then germination was monitored every day.
3. Results 3.1. Exogenous H2O2 enhanced seed germination and seedling growth of both vegetables Coriander and carrot seeds were treated with several concentrations of H2O2 solution under room temperature. The pH of all H2O2 solutions before and after soaking seeds was between 5.43 and 5.59. Fig. 1A shows the percentage of coriander seed germination after treatment with H2O2 for 12 and 24 h. The percentage of seed germination was significantly increased after treatment with all concentrations of H2O2 solutions. Particularly, 25 mM H2O2 solution showed the greatest effect; 82 and 87% germination after 12 and 24 h, respectively. In the treatment with 200 mM H2O2 solution, the percentage of seed germination was initially reduced but became higher than that of control (no H2O2) after 120 h. Fig. 1B shows the morphology of germinated coriander seeds. Radicle of embryo was emerged through seed coat earlier in seeds treated for 12 h than 24 h, Germination index was also
2.3. Seed germination and seedling growth analysis For analysis of coriander and carrot seed germination, we counted number of germinated seeds every 24 h. Germinated seed was determined by the emergence of the radicle from seed coat. Seed germination rate was calculated as follows; 86
Scientia Horticulturae 227 (2018) 85–91
K. Panngom et al.
Fig. 1. The effect of H2O2 on seed germination of coriander. (A) The percentage of seed germination after treatment with H2O2; 25, 50, 100 and 200 mM for 12 and 24 h. (B) The morphology of germinated coriander seeds after 48 and 96 h. (C) Germination index (GI) of coriander seeds. *p < 0.05 or **p < 0.01.
(Fig. 2A). Differently from coriander seeds, carrot seeds germinated faster in the treatment for 24 h than 12 h as shown in Fig. 2A and B. Radicles of carrot seeds treated for 24 h were emerged from seed coat earlier and longer, compared to the 12 h treated seeds (Fig. 2B). Moreover, germination index of carrot seeds was significantly increased after treatment with 25 and 50 mM H2O2 for 24 h (Fig. 2C). These results indicate that carrot seeds require higher concentration and longer treatment of H2O2 than coriander seeds.
significantly increased in coriander seeds treated with H2O2 solutions with the highest effect in 25–50 mM (Fig. 1C). For carrot seeds treated with H2O2 solutions, the percentage of seed germination was significantly increased and 25 and 50 mM concentrations resulted in the highest effect in both 12 and 24 h treatment (Fig. 2A). The percentage of germination was up to 71% in the treatment for 24 h (Fig. 2A). Increase in germination percentage was slowed down when the concentration of H2O2 was higher than 50 mM
Fig. 2. The effect of H2O2 on seed germination of carrot. (A) The percentage of seed germination after treatment with H2O2; 25, 50, 100 and 200 mM for 12 and 24 h. (B) The morphology of germinated carrot seeds after 24 and 48 h. (C) Germination index (GI) of carrot seeds. *p < 0.05 or **p < 0.01.
87
Scientia Horticulturae 227 (2018) 85–91
K. Panngom et al.
Seedling growth was analyzed by measuring the length of seedlings after 5 (carrot) and 6 (coriander) days. The seedling length of coriander was significantly increased after H2O2 treatment for both 12 and 24 h although the increase was slower in higher concentration of H2O2 and 24 h treatment (Fig. 3A). For carrot plant, the seedling length was also increased significantly after H2O2 treatment and there was no significant difference among H2O2 concentration and treatment time (Fig. 3B). 3.2. Exogenous NO enhances seed germination and seedling growth of coriander more greatly than carrot In order to compare the effects between ROS and RNS on seed germination and seedling growth, we treated coriander and carrot seeds with nitric oxide (NO) generated from SNP (sodium nitroprusside). The pH of SNP solutions before and after soaking seeds was between 5.18 and 5.35. Germination percentage of coriander seeds was significantly increased after SNP treatment and the increase was faster in 12.5 μM treatment for both 12 (79%) and 24 (84.5%) hrs (Fig. 4A). The radicle emergence from seed coat was slightly earlier in 12 h treatment (Fig. 4B). Germination index of coriander seeds treated with SNP was significantly higher than control in both 12 and 24 h treatment (Fig. 4C). Carrot seeds treated with SNP exhibited no dramatic changes in germination percentage compared to control although increased germination was observed in the treatments with several concentrations of SNP (Fig. 5A and B). Generally, germination level was slightly lower in 24 than 12 h treatment (Fig. 5A). Germination index of SNP treated seeds was not significantly different from that of control in both 12 and 24 h treatment (Fig. 5C). These results demonstrate that carrot seeds seem to respond more sensitively to H2O2 than NO generated from SNP in terms of seed germination. To determine the effect of RNS on plant growth, we assessed the influence of SNP treatment on seedling growth by measuring the length. The length of coriander seedlings germinated from seeds treated with SNP was significantly increased with the greater effect in 12.5 and 25 μM treatment for 12 h (Fig. 6A). In case of carrot, seedlings length was longer in SNP treatment for 12 h than in control even though seed germination was not significantly affected by SNP treatment (Fig. 6B). Longer radicles were observed in both coriander and carrot plants in the treatments with 12.5 and 25 μM SNP for 12 h (Fig. 6A and B). In addition, seedling growth was more dramatically increased in coriander than carrot in response to SNP treatment. 3.3. H2O2 and NO treatment can modulate seed metabolisms during germination To elucidate the distinctive effect of RONS on seed metabolisms during germination, we measured the level of total soluble proteins and reducing sugar (α-amylase activity indicator) in germinating seeds at 0 and 72 h after treatment with H2O2 and SNP for 24 h. Amounts of total soluble proteins in coriander and carrot seeds after H2O2 treatment were significantly increased (Fig. 7A). After 72 h, protein level in coriander seeds was slightly reduced except 25 mM treatment (Fig. 7A). However, carrot showed the increased level of soluble proteins even after 72 h (Fig. 7A). In SNP treatment, concentration of total soluble proteins was significantly increased in coriander, especially in 12.5 and 25 μM SNP treatment (Fig. 7B). In carrot, no significant change in protein level was observed in all treatments (Fig. 7B). The quantity of reducing sugar in coriander seeds was significantly increased at 72 h post germination in treatments with 50 and 100 mM H2O2 solutions (Fig. 8A). In carrot, higher level of reducing sugar was observed in treatments with most H2O2 concentrations and in both 0 and 72 h (Fig. 8A). However, treatment with NO generated from SNP did not cause any change in the level of reducing sugar in both coriander and carrot (Fig. 8B). These results suggest that seed metabolisms
Fig. 3. The length of coriander and carrot seedlings grown from seeds treated with H2O2 for 6 days. (A) The length and morphology of coriander seedlings. (B) The length and morphology of carrot seedlings. *p < 0.05 or **p < 0.01.
88
Scientia Horticulturae 227 (2018) 85–91
K. Panngom et al.
Fig. 4. The effect of NO generated from SNP on germination of coriander seeds. (A) The percentage of seed germination after treatment with NO generated from SNP in concentrations; 12.5, 25, 50 and 100 μM for 12 and 24 h. (B) The morphology of germinated coriander seeds after 48 and 96 h. (C) Germination index (GI) of coriander seeds. *p < 0.05 or **p < 0.01.
plant species. In our results, exogenously applied H2O2 solutions can increase the efficiency of seed germination and subsequent seedling development in both coriander and carrot. However, NO generated from SNP showed greater activation effect on coriander than carrot. Carrot exhibited the reduced level of H2O2 effect on germination and growth compared to coriander. This may be because carrot has higher scavenging or antioxidant activity for H2O2 and NO than coriander.
can be differently affected by exogenous RONS depending on plant species and individual reactive species. 4. Discussion Although RONS are known as signaling molecules in various cellular processes, our results suggest that RONS can act differently in different
Fig. 5. The effect of NO generated from SNP on germination of carrot seeds. (A) The percentage of seed germination after treatment with NO generated from SNP in concentrations; 12.5, 25, 50 and 100 μM for 12 and 24 h. (B) The morphology of germinated carrot seeds after 24 and 48 h. (C) Germination index (GI) of carrot seeds. *p < 0.05 or **p < 0.01.
89
Scientia Horticulturae 227 (2018) 85–91
K. Panngom et al.
Fig. 7. The amount of total soluble proteins in germinated coriander and carrot seeds treated with H2O2 and NO generated from SNP (24 h) after 0 and 72 h post-germination. *p < 0.05 or **p < 0.01.
Thus, treatment with more H2O2 and NO may be required to achieve similar level of developmental activation in carrot. Possible functions of H2O2 and NO during germination have been suggested (Barba-Espin et al., 2010; Barba-Espin et al., 2012; Liu et al., 2010; Kopyra and Gwozdz, 2003). H2O2 is known to induce ABA catabolism and GA synthesis and this may stimulate early seed germination in plants (Barba-Espin et al., 2010; Barba-Espin et al., 2012; Liu et al., 2010). NO stimulates the activity of α-amylase during germination (Kopyra and Gwozdz, 2003). In our study, the level of reducing sugar, an indicator for α-amylase activity, in germinating seeds was not affected by NO treatment although H2O2 treatment promoted the generation of reducing sugar in both coriander and carrot. H2O2 treatment may enhance the activity of α-amylase probably through GA hormone induction in both coriander and carrot. However, increase in the level of reducing sugar was more dramatic in carrot than coriander although germination was more greatly enhanced by H2O2 in coriander. This may be because data of reducing sugar amount is not enough to assess the activity of α-amylase or another mechanism(s) is possibly involved in H2O2 action. About action mechanism(s) of NO during seed germination, it seems to be various depending on plant species. In our study, carrot is not affected by NO treatment. In addition, the amount of reducing sugar was not changed after NO treatment in both coriander and carrot. Since NO is a well-known regulator for abiotic and biotic stresses, the influence of NO on plant development may demonstrate broad spectrum of outcomes in various plant species. From our study, it was not clearly known how NO could stimulate germination of coriander seeds. Besides activation of α-amylase, another mechanism(s)
Fig. 6. The length of coriander and carrot seedlings grown from seeds treated with NO generated from SNP for 6 days. (A) The length and morphology of coriander seedlings. (B) The length and morphology of carrot seedlings. *p < 0.05 or **p < 0.01.
90
Scientia Horticulturae 227 (2018) 85–91
K. Panngom et al.
and seedling growth in carrot while NO generated from SNP did not cause any significant change. Differential effects on plant development were generated depending on plant species and dose of individual reactive species. Our results provide useful information in understanding comparative influence of two reactive species on early development of vegetable species. Conflict of interest The authors declare no conflict of interest. Acknowledgment This work was partially supported by the grants project from Maejo University Phrae Campus in the year of 2015 (MJ.2-58-005). References Arc, E., Galland, M., Godin, B., Cueff, G., Rajjou, L., 2013. Nitric oxide implication in the control of seed dormancy and germination. Front. Plant Sci. 4, 346. Barba-Espin, G., Diaz-Vivancos, P., Clemente-Moreno, M.J., Albacete, A., Faize, L., Faize, M., Perez-Alfocea, F., Hernandez, J.A., 2010. Interaction between hydrogen peroxide and plant hormones during germination and the early growth of pea seedlings. Plant Cell Environ. 33, 981–994. Barba-Espin, G., Hernandez, J.A., Diaz-Vivancos, P., 2012. Role of H2O2 in pea seed germination. Plant Signal. Behav. 7 (2), 193–195. Bewley, J.D., 1997. Seed germination and dormancy. The Plant Cell 9, 1055–1066. Cavusoglu, K., Kabar, K., 2010. Effects of hydrogen peroxide on the germination and early seedling growth of barley under NaCl and high temperature stresses. Eurasia J. Biosci. 4, 70–79. Gallardo, K., Job, C., Groot, S.P.C., Puype, M., Demol, H., Vandekerckhove, J., Job, D., 2001. Proteomic analysis of Arabidopsis seed germination and priming. Plant Physiol. 126, 835–884. Gondim, F.A., Gomes-Filho, E., Lacerda, C.F., Prisco, J.T., Azevedo Neto, A.D., Marques, E.C., 2010. Pretreatment with H2O2 in maize seeds: effects on germination and seedling acclimation to salt stress. Braz. J. Plant Physiol. 22 (2), 103–112. Graves, D.B., 2012. The emerging role of reactive oxygen and nitrogen species in redox biology and some implications for plasma applications to medicine and biology. Appl. Phys. 45, 263001. Gutterman, Y., 1994. Strategies of seed dispersal and germination in plants inhabiting deserts. Bot. Rev. 60, 373. Ji, S.H., Kim, T., Panngom, K., Hong, Y.J., Pengkit, A., Park, D.H., Kang, M.H., Lee, S.H., Im, J.S., Kim, J.S., Uhm, H.S., Choi, E.H., Park, G., 2015. Assessment of the effects of nitrogen plasma and plasma-generated nitric oxide on early development of Coriandum sativum. Plasma Process. Polym. 12, 1164–1173. Koger, C.H., Reddy, K.N., Poston, D.H., 2004. Factors affecting seed germination, seedling emergence and survival of Texas weed (Caperonia palustris). Weed Sci. 52, 989–995. Kopyra, M., Gwozdz, E.A., 2003. Nitric oxide stimulates seed germination and counteracts the inhibitory effect of heavy metals and salinity on root growth of Lupinus luteus. Plant Physiol. Biochem. 41, 1011–1017. Laribi, B., Kouki, K., Hamdi, M., Bettaieb, T., 2015. Coriander (Coriandrum sativum L.) and its bioactive constituents. Fitoterapia 103, 9–26. Liu, H.Y., Yu, X., Cui, D.Y., Sun, M.H., Sun, W.N., Tang, Z.C., Kwak, S.S., Su, W.A., 2007. The role of water channel proteins and nitric oxide signaling in rice seed germination. Cell Res. 17, 638–649. Liu, Y., Ye, N., Liu, R., Chen, M., Zhang, J., 2010. H2O2 mediates the regulation of ABA catabolism and GA biosynthesis in Arabidopsis seed dormancy and germination. J. Exp. Bot. 61 (11), 2979–2990. Miransari, M., Smith, D.L., 2014. Plant hormones and seed germination. Environ. Exp. Bot. 99, 110–121. Msaada, K., Jemia, M.B., Salem, N., Bachrouch, O., Sriti, J., Tammar, S., Bettaieb, I., Jabri, I., Kefi, S., Limam, F., Marzouk, B., 2017. Antioxidant activity of methanolic extracts from three coriander (Coriandrum sativum L.) fruit varieties. Arab. J. Chem. 10 (2), S3176–S3183. Panuccio, M.R., Jacobsen, S.E., Akhtar, S.S., Muscolo, A., 2014. Effect of saline water on seed germination and early seedling growth of the halophyte quinoa. AoB PLANTS 6, plu047. Pereira, R.S., Nascimento, W.M., Vieira, J.V., 2008. Carrot seed germination and vigor in response to temperature and umbel orders. Sci. Agric. 65 (2), 145–150. Rithichai, P., Sampantharat, P., Jirakiattikul, Y., 2009. Coriander (Coriandrum sativum L.) seed quality as affected by accelerated aging and subsequent hydropriming. Asian J. Food Agro-Ind. (Special Issue), S217–S221. Silva Dias, J.C., 2014. Guiding strategies for breeding vegetable cultivars. Agric. Sci. 5, 9–32. Weitbrecht, K., Müller, K., Leubner-Metzger, G., 2011. First off the mark: early seed germination. J. Exp. Bot. 62 (10), 3289–3309.
Fig. 8. The amounts of reducing sugar in germinated coriander and carrot seeds treated with H2O2 and NO generated from SNP (24 h) after 0 and 72 h post-germination. *p < 0.05 or **p < 0.01.
may be possibly involved, but further intense investigation will be needed. Our results demonstrate that H2O2 and NO can activate biochemical changes in seeds during germination. Biochemical changes during seed germination are well demonstrated in studies; during seed imbibition, metabolisms of stored proteins (especially enzymes) can be activated in seeds and also energy-related enzymes for major metabolic pathways play important roles in inducing early seed germination in plant (Weitbrecht et al., 2011). The level of total soluble proteins in germinating carrot and coriander seeds is significantly increased after H2O2 treatment. NO treatment elevates the amount of total soluble proteins in coriander but not in carrot. These results suggest that gene expression inside coriander and carrot seeds is stimulated by exogenous H2O2 and NO even though carrot responds to NO less sensitively. It may be possible that NO level used in the treatment is not enough to induce molecular and developmental activation in carrot. 5. Conclusion Effects of exogenous H2O2 and NO generated from SNP on seed germination, growth and development of seedlings, and seed metabolisms during germination were analyzed in coriander and carrot. Both H2O2 and NO induced enhancement in germination and seedling growth in coriander. However, only H2O2 promoted seed germination
91