PHYSIOLOGICAL AND ENZYMATIC CHANGES DURING SEED MATURATION AND GERMINATION OF Luehea divaricata

Luehea divaricata Mart. & Zucc is tree is of great ecological and economic importance. This study investigated the effect of fruit maturation on the germination and vigor of L. divaricata. Fruits were harvested at three maturity stages (unripe or green, brown and open) defined by digital colorimetry and seeds extracted from each stage divided into lots of earliest harvested and stored. Germination test was conducted with four replicates of 25 seeds from each fruit maturity stage in plastic boxes under temperature of 30 ± 2° C. The study was conducted in a 2 x 3 (two lots and three maturity stages) factorial arrangement in a completely randomized design with four replications. Data was submitted to analysis of variance and the means were compared by Tukey at 5%. Germination, germination speed index, average root length and moisture content showed higher values for recently harvested seeds extracted from fruits of all maturity stages compared to stored seeds. LEA protein showed higher activity in seeds from fruits with brown epicarp color of both lots while α-amylase protein activity was higher in the stored seed lot. This shows that better seeds are those that are newly collected fruits and browns.

In order to obtain high quality seeds it is necessary to pinpoint physiological maturation of both fruits and seeds which is not yet well documented in the production of reproductive structure of L.divaricata.
Physiological maturity of fruits is important for determination of the ideal period for harvest, processing, drying, storage and quality control of seeds (AGUIAR et al., 2007).Seed physiological maturity is when seeds reach maximum physiological quality, vigor, germination, size and dry matter weight.At that stage, degenerative changes begin compromising germination and vigor (CARVALHO;NAKAGAWA, 2012).Seed physiological maturity is importance for definition of the ideal time to collect seeds with high physiological quality.
For determination of the physiological maturity in seeds, several fruit characteristics are used such as changes of fruit epicarp color (VIDIGAL, 2008;DIAS et al., 2006), fruit size and seed weight (DIAS et al., 2006;COSTA et al., 2006), and seed moisture content (ARAÚJO et al., 2006).In light of the above arguments, this essay aimed to determine changes during fruit maturation on germination and vigor of L.divaricata seeds.

MATERIALS AND METHODS
We collected fruits of L. divaricata from seven mature trees located in the western region Parana state, Brazil, at three stages based on fruit epicarp color: green, brown, and ripe and open fruits.Seeds were manually extracted after collection.
Seeds from each fruit stage were divided into two lots: one formed by fresh collected seeds, while the other lot was formed with seeds stored for 30 days in an uncontrolled environment room.
Fruit mean diameter and length were measured with a digital caliper.Seed moisture content (wet basis) was determined in the laboratory by oven method at 105±3° C for 24 hours (BRASIL, 2009) with four replicates of 100 seeds from each maturity stage and seed lot.
Germination test used four replicates of 25 seeds from each maturity stage.Seeds were sown using Germitest paper, moistened with 2.5 times its weight with distilled water (BRASIL, 2009) inside plastic boxes placed in germination chamber at 30±2° C, with a photoperiod of 12 hours of light.
Measurement of germination were made daily, with normal germinated seedlings being considered as those that presented a developed root system.Data were expressed as percentage of normal germinated seedlings.At the same time, the germination speed index (GSI) was calculated according to Maguire (1962).
Average root length (ARL) was determined with a ruler.For seedling dry matter (SLDM), ten seedlings were taken at random from the substrate, placed in an oven at 65º C for 48 hours for weighing (±0.0001g).
Evaluation of epicarp color was performed by digital colorimetry (ACR-1023 by Instrutherm®) on 25 L. divaricata fruits from each maturity stage and reproduced with a software (Photoshop CS6) producing the colors represented in figure 1.Samples of 1.5 grams of seeds from fruits of each maturity stage were homogenized in 4 mL of 0.01 M sodium phosphate buffer at pH 6.0 (extraction buffer) in a previously cooled porcelain mortar with the addition of 0.04 g of polyvinylpyrrolidone (PVP) during maceration.The homogenized product was centrifuged at 20.000 g for 20 minutes.The supernatant obtained, considered, as the fraction containing soluble proteins was stored at 4 o C for later biochemical analyses (LUSSO and PASCHOLATI, 1999).
Activity of α-amylase enzyme and LEA protein were determined using methodology described by Ching (1973).For determination of the α-amylase, 1 mL of starch solution was added to the extract, and the subsamples placed in an incubator at 30° C.After 5 minutes, 1 mL of I 2 +KI and 9 mL of distilled water were added.
Spectrophotometer readings at 620 nm for used 1 mL of buffer solution plus 9 mL of distilled water as blank and the substrate was composed by 1 mL of starch solution plus 1 mL of I 2 +KI plus 9 mL of distilled water were made.Total activity of α-amylase enzyme was calculated by {[(substrate) x 620 -(sample) x 620]/5X} x 20 extraction/(0.1aliquotx 1.5 grams of seeds), where X=1µg, while starch = [(substrate) x 620]/1000 with the results being expressed as µg of hydrolyzed starch min -1 per seed.
For determination of the LEA protein, 1 mL of T-med solution (bovine albumin, copper sulfate and sodium hydroxide) was added to the extract, and the sub-samples placed in an incubator at 30° C.After 5 minutes, 1 mL of I 2 +KI and 9 mL of distilled water were added.
Spectrophotometer readings at 525 nm for LEA protein used 1 mL of buffer solution plus 9 mL of distilled water as blank and the substrate was composed by 1 mL of starch solution plus 1 mL of I 2 +KI plus 9 mL of distilled water were made.The activity of the LEA protein was given by Ct=FC x At, where Ct = test concentration, FC = calibration factor of the LEA protein, At = test absorbance with data expressed in g dL -1 x 10.
The essay was conducted as factorial (2 x 3) composed by two seed lots and three fruit maturity stages with four replications.Normal distribution of the data was evaluated by the Lillifords test, which presented normal distribution.The mean values were compared by the Tukey test at 5% probability.Pearson simple correlation analysis was also performed between the results of the enzyme analysis tests and the germination tests using Genes software (CRUZ, 2006).

RESULTS AND DISCUSSION
Length of L. divaricata fruits (Table 1) did not result in differences (P>0.05) as a function of maturity stages.This result occurs after the fruit reaches maximum size.Reduction of fruit size depends on the species and on the degree of dehydration found by the end of fruit maturation (MARCOS FILHO, 2005) 1).Similar trend was observed by Alves et al. (2005) in fruits from Mimosa caesalpiniifolia Benth.which showed a gradual increase in length and width throughout the maturation process.However, those authors reported that fruit visual index was not an effective predictor of physiological seed maturity.

Stages of maturation Length Mean diameter
The greatest values for seed moisture content were obtained from the fresh collected seed lot of fruits classified as green and brown (Table 2).Even so, those values were smaller than those measured by Marini et al. (2012) with L.grandiflora seeds that exhibited 13% moisture in the black maturity stage.
Seeds from L. divaricata fruits classified as green and open/ripe exhibited greater germination values than from brown fruits.No differences (P>0.05) were detected between the fresh and the stored seed lots.In an analogous way, vigor evaluated by the GSI with seeds from unripe fruits exhibited the greatest values, which differed from those of brown fruits, without difference between the two seed lots (Table 2).
The highest ARL was measured in seedlings obtained from seeds of green and recently harvested fruits compared to those from seedlings produced from seeds of either brown or open/ripe fruit stages as well as stored seed lot (Table 3).ARL was synchronized with GSI and germination percentage, indicating that seeds from green fruits showed greater vigor and germination potential.Therefore, ARL and fruit epicarp color are good indicators of physiological maturity of L. divaricata seeds.
In addition, Oro et al. (2012) studying maturity stages of cerejeira-do-mato (Eugenia involucrata DC.) fruits observed that high germination speed were recorded with seeds from clear red fruits.Those authors stated that seeds from that fruit development stage showed greater vigor similarly with the results in the present essay.108 FLORESTA, Curitiba, PR, v. 47, n. 1, p. 105 -111, jan. / mar. 2017. Schulz, D. G. et  Seed lot influenced (P<0.05)seedling dry matter.Highest dry matter values were recorded in seedlings from the recently collected seed lot.No differences (P>0.05)resulted as result of fruit maturity stages.
On the other hand, seed dry matter (SDM) was higher from seeds of green fruits from both lots.However, seeds from the recently collected seed lot resulted in a greater accumulation of dry matter when compared to stored seeds from unripe fruits (Table 3).Table 3.Average root length (ARL), seedling dry matter (SLDM) and seed dry matter (SDM) in L. divaricata seed lots from fruits harvested as a function of maturity stages, 2012.Tabela 3. Comprimento médio de raiz, massa seca de plântulas, e matéria seca de sementes de L. divaricata recem colhidas e armazenadas em diferentes estádios de maturação do fruto, 2012.During seed germination, α-amylase plays an important role in starch hydrolysis in metabolizable sugars (LIMA et al., 2008).Activity of α-amylase is of fundamental importance in seed germination because of the effects from high temperatures during seed drying (ROSA et al., 2005).The lack of synchronism between expressiveness of α-amylase and seed vigor is due to the inactivation of the germination process in the seeds used for the test resulting in unreliable data for isolated use of the enzymatic activity test of α-amylase as a vigor index in studies of L. divaricata seeds.
The Pearson correlation between percentage and speed rate of germination was significant and positive.On the contrary, correlation between germination percentage and GSI showed high negative values with LEA protein indicating that the greater the values of germination percentage and GSI the lower the values of LEA protein in the seeds.The other variables were independent (Table 4).Meneses et al. (2006) reported that LEA proteins were described on genes abundantly expressed during final stage of desiccation in seed development, thus showing the negative correlation with germination percentage and GSI, which were greater in L. divaricata seeds with higher moisture content (Table 2).

CONCLUSION
 L. divaricata seeds should be harvested from fruits with green epicarp (R=94, G=76 and B=49). Germination percentage, GSI, ARL, SDM, seed moisture content, fruit color and LEA protein are indicators of physiological maturity of L. divaricata seeds. Germination percentage, GSI, ARL, SDM, and seed moisture content showed the highest values in seeds from green fruits from the recently collected seed lot at all stages of physiological maturity. LEA protein showed greater activity in seeds obtained from fruits desplaying brown epicarp from both seed lots. The α-amylase enzyme showed greater activity in seeds from the stored seed lot.

Figure 2 .
Figure 2. Activity of α-amylase and enzymes LEA protein from two L. divaricata seed lots as a function of fruit maturity stages -1: Green; 2: Brown; 3: Open/ripe.Columns followed by the same lowercase letter for the different stages of maturation and upper case for the different seed lots not differ among themselves by the Tukey test at 5% probability.Figura 2. Atividade da α-amilase e protein LEA em dois lotes de sementes de L. divaricata em função do estádio de maturação.1: verde; 2: marron; 3: aberto.
. Different results were reported by Marini et al. (2011) with Luehea grandiflora Mart.& Zucc where fruits from the open stage exhibited the smallest lengths.Fruits of L. divaricata classified as at the open/ripe stage exhibited greater diameter values (9.70 mm) compared to those classified as green and brown (Table

Table 2 .
Germination percentage (%G), germination speed index (GSI) and moisture content in seed lots coming from fruit harvested at three maturity stages, 2012.Tabela 2. Porcentagem de germinação (%), índice de velocidade de germinação e grau de umidade em sementes de L. divaricata recem colhidas e armazenadas em diferentes estádios de maturação do fruto, 2012.Mean values followed by the same small letter in the column and capital letter in the line do not differ among themselves by the Tukey test at 5% probability.CV= Coefficient of variation.DMS: Minimum significant difference.
Mean values followed by the same small letter in the column and capital letter in the line do not differ among themselves by the Tukey test at 5% probability.CV= Coefficient of variation.DMS: Minimum significant difference.
* Significant at 5% and ** at 1% probability of error by the T test.ns Not significant.b