Expression of Enzymes During the Germination of Seeds in Endangered Cerrado Species

The objective of this study was to evaluate the enzymes expression during the seeds germination process of ever-lasting species Comanthera elegans and Comanthera bisulcata. For the evaluation of the seeds physiological potential, the germination test and index of germination speed were performed. The expression of enzymes esterase (EST), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), superoxide dismutase (SOD), catalase (CAT) and endo-β-mannanase during the germination process were evaluated. The expression of these enzymes was evaluated in dried seeds, in the protrusion, in the emergence of the primal leaf, at the beginning of the formation of normal seedling and dormant seeds at the end of the germination process. To the extent that the germination process occurs in the species C. bisulcata and C. elegans there is greater expression of the enzyme CAT and lower of the enzyme EST. There is variation in the expression of the enzymes SOD, ADH and MDH in seeds of both species during the germination process. The enzyme endo-β-mannanase presents greater activity in seeds with radicle protrusion in the two studied species.


Introduction
The plants known as everlasting, of the species Comanthera elegans (Bong.)Parra and Giul and Comanthera bisulcata (Koern.)L.R.Parra & Giul, belong to the Eriocaulaceae family (Parra et al., 2010).Of natural occurrence in Brazil in the rupestrian fields, mainly in the region of Diamantina, MG, they have great commercial interest for its population, due to the fact of the flowers to be harvested, to conserve the color and shape for a long period.Due to the disorderly extractivism by presenting high value mainly in the international market, the roots are pulled up along with the scapes and flowers before the seeds formation, thus leaving a minimum number of individuals in the areas, which makes it inviable not only their regeneration but also the production of new seeds.Some species of this genus, as for example the Comanthera elegans, are included in the list of endangered species (Brazil, 2014).Thus, it becomes necessary to carry out measures to guarantee their conservation.
There is a great interest in the study on the livelihood strategies of everlasting plants, which comprise since the seeds development until the formation of the adult plant.Therefore, it is of fundamental importance the knowledge of the germination process in which metabolic changes occur in the embryo until the seedling formation.In addition to extrinsic factors related to germination, the intrinsic ones must be also considered as the enzymes expression during this process.
Several authors have observed low physiological quality in everlasting seeds.Oliveira et al. (2015) affirm that the highest rate of germination of C. elegans, at a temperature of 25±2 °C, was 22.932% for seeds collected in the month of September.In the collections in the periods that precede or follow this month, the percentage of germination did not surpass 11.72%.Still the same authors affirm that the germination results vary with the day of harvest, with the site and with the flowering.There is, however, the need to study the causes which observed a low physiological quality, which can be elucidated by means of studies of enzyme expression.
In the case of seeds, the study of enzyme expression has helped in the understanding of the biochemical processes that are associated with their quality.As an example, one may mention the enzymes that participate in the reactions of lipid oxidation as the esterases (Rejon et al., 2012).There are also those who act in breathing as the malate dehydrogenase and alcohol dehydrogenase (Tunes et al., 2011) and even the enzymes with antioxidant function as the catalase and superoxide dismutase (Wang et al., 2015).Thus, identifying the enzymes that are involved in the metabolic reactions responsible for the synthesis and degradation of molecules, can provide subsidies to understand the mechanisms involved in seed germination, as the activities related to the biochemical processes.
In this context, the objective of this study was to evaluate the enzymes expression during the germination process of ever-living species seeds Comanthera elegans and Comanthera Bisulcata, using the electrophoresis technique.

Material and Methods
The collection of the inflorescences of ever-living plants (Comanthera elegans and Comanthera bisulcata) was performed in the city of Diamantina, MG between the months of May to August 2015 at representative locations of the production area.
For the seeds extraction inflorescences were selected with the entire flower head opened that were crushed in a blender for 30 seconds, removing all inert material with the aid of sieves to obtain the pure seed.The determination of water content was obtained by the oven method at 130 °C for 1 hour, using two replications of 0.1 g for each species (MAPA, 2009).The germination test was conducted with four replications of 100 seeds, with sowing in germination blotter paper soaked with distilled water in a proportion of 2.5 times the weight of the paper and placed in gerbox-type plastic boxes.The seeds remained in BOD during 35 days in alternating temperature of 25 °C/16 hours in the presence of light and 15 °C/8 hours in the dark for the species Comanthera elegans according to Pereira et al. (2014) and to Comanthera bisulcata was 20 °C/16 hours in the presence of light and 15 °C/8 hours in the dark according to Oliveira and Garcia (2011).The results were expressed as mean percentage of normal seedlings.For the seeds remaining from the germination test, the tetrazolium test was performed for the determination of its viability in a solution containing 0.5% of 2, 3, 5 triphenyl tetrazolium chloride for 3 hours, in the dark, at a temperature of 30 °C.The evaluation was performed with the aid of stereoscopic microscope.
The germination speed index was performed together with the germination test, by daily counts of the number of seeds with radicle protrusion until the end of the germination test and index calculated according to Maguire (1962).
The evaluation of the enzyme expression was carried out during the germination process by means of the technique of electrophoresis.To obtain seeds and normal seedlings at different stages of the germination process, these were sown on paper substrate and classified as: (1) dry seed (S), (2) beginning of the protrusion (Pro), (3) emergence of the primary leaf (FP), (4) beginning of the formation of normal seedlings (Plt) and ( 5) dormant seeds at the end of the germination process (SD).
The obtaining of seed and the normal seedlings in the 4 phases were respectively at 6, 9, 20 and 35 days after soaking for C. elegans and 9, 13, 25 and 35 days for C. bisulcata.To obtain a total volume of 100 mg of seeds and seedlings, several sowings were carried out.The seeds and normal seedlings were stored in deep freezer (-86 C) for the electrophoretic analyzes.
For the extraction of enzymes 100 mg of seeds and seedlings were macerated in the presence of polyvinylpyrrolidone (PVP) and liquid nitrogen.250 µl of extraction buffer were added (Tris-HCl, pH 8.0) and 0.1% of β-mercaptaenol, were maintained "overnight" and then centrifuged at 14000 rpm for 30 minutes at 4 °C.Out of the supernatant 60 μL was removed with subsequent application in polyacrylamide gel 7.5% (separator gel) and 4.5% (GEL CONCENTRADOR).The gels were revealed according to the methodology described by Alfenas (2006).
For analysis of the enzymatic activity of endo-β-mannanase in each 100 mg of powder of each sample 300 µL of extraction buffer were added [0.1 M Hepes/0.5 M NaCl and ascorbic acid (5 mg of ascorbic acid per ml of buffer, pH 8.0].The samples were centrifuged for 30 minutes at 14000 rpm and 2 µL of supernatant applied in gel containing 6 mL of LBG(locust bean gum), 0.24 g of agarose and 24 mL of buffer pH 5.0 (1 M Citric Acid/0.4M of Na 2 HPO 4 •2H 2 O).The aliquots were applied in 2 mm holes made in the gel using an awl.The gel was incubated for 21 h and revealed according to the methodology proposed by Silva et al. (2004).The measurement of the halos diameter in two directions was performed with the aid of the Software ImageJ ® resulting in an average and a comparison was made with the standard curve generated by endo-β-mannanase enzyme of Aspergillus niger (Megazyme).The calculation of the activity of the enzyme endo-β-mannanase was performed according to Downie et al. (1994).
The interpretation of the results was based on a qualitative analysis of the electrophoresis gels, considering the presence/absence, as well as the intensity of each one of the electrophoretic bands in each iso-enzymatic system evaluated.For the quantitative analysis of the bands the image analysis Software ImageJ (Rasband, 1997) was used measured in pixel 2 .This software has been specially developed to measure the isoforms contained in each of the trenches.

Results and Discussion
The water content of the seeds of the species C. bisulcata and C. elegans were 9% and 7%, indicating that they are richer in oil seeds, as they reach the point of hygroscopic equilibrium with lower levels of water, under temperature conditions of 25 C and relative humidity of 60%.Some authors have observed the same behavior in oilseeds as Antoniassi et al. (2013) and sesame, Faugno, et al. (2016).
According to the results in Table 1, variation is observed between the two species for the evaluated physiological quality by means of the germination test and GVI.Superior values of germination and GVI were observed in seeds of the species C. elegans comparing to C. bisulcata.For the results of the tetrazolium test at the end of the germination test, it was observed that the remaining seeds were not viable (Table 1).Similar results regarding the low percentage of germination in everlasting seeds were found in the work of Garcia et al. (2014); Oliveira et al. (2015).These low germination results are expected since many authors have highlighted some aspects such as: climate variation that can influence the beginning and end of the harvest, the physiological state in which the seed is, the period of maturation or could be by climatic conditions and/or edaphic micro conditions of the place in where the plants developed in which seeds were produced (Oliveira & Garcia, 2011).Oliveira et al. (2015) while studying the collection period of C. elegans, stated that collections before May, the seeds germination rates were below 50%.These results agree with those found in the literature and prove that the collection of inflorescences occurs before the period of seeds formation and that only a small portion of these can achieve the physiological maturity.
By electrophoretic analysis of the enzyme esterase (EST) (Figure 1), a higher expression was observed in dried seeds with small concentration differences in the two species (1643 and 1547 pixel 2 ).For C. bisulcata there was a gradual decrease of EST along the germinative process and for the species C. elegans a more pronounced decrease was observed.

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Table 1 .
Average values of hard seeds, dormant, dead and of normal seedlings obtained from the germination test (%), and germination speed index (GSI) of everlasting seeds