D.S. VIDIGAL, D.C.F.S. DIAS, E.R.V. VON PINHO AND L.A.S. DIAS
Vidigal, D.S., Dias, D.C.F.S., Von Pinho, E.R.V. and Dias, L.A.S. (2009), Seed Sci. & Technol., 37, 192-201
Sweet pepper seed quality and lea-protein activity in relation to fruit maturation and post-harvest storage D.S. VIDIGAL1, D.C.F.S. DIAS1*, E.R.V. VON PINHO2 AND L.A.S. DIAS1 1
Department of Fitotecnia, University Federal of Viçosa, 36570-000, Viçosa, MG, Brazil (E-mail:
[email protected]) 2 Department of Agriculture, University Federal of Lavras, 37200-000, Lavras, MG, Brazil
(Accepted July 2008)
Summary This study was conducted to evaluate the influence of fruit maturity and post-harvest storage on physiological quality and LEA proteins activity of sweet pepper (Capsicum annuum L.) seeds. Fruits were harvested at 40, 50, 60 and 70 days after anthesis (DAA) and stored for 0, 3, 6, 9, 12 and 15 days before seed extraction. Seeds were evaluated by germination, first count, seedling length, accelerated aging, speed emergence index, electrical conductivity and eletrophoretical analysis for proteins LEA. It was verified that precocious harvest (40 DAA) was not beneficial to physiological quality of pepper seeds even when associated to 15 days post-harvest storage. For fruits harvested at 50 DAA, 12 days post-harvest storage was indispensable to assure the physiological quality of seeds. Pepper seeds harvested from 60 DAA presented high quality, not being necessary the postharvest storage of fruits. LEA proteins synthesis occurred at 60 DAA, being directly related to physiological quality of pepper seeds.
Introduction The studies regarding seed maturation have contributed significantly to determine the ideal fruit development stage for harvesting to obtain high quality seeds. Because of continuous flowering and fruiting in indeterminate growth plants, such as sweet pepper, fruits of different physiological maturity are found on the same plant, making it difficult to define the most adequate fruit harvest time. During development, the maximum seed germination and vigour may not coincide with the maximum dry matter accumulation that characterizes physiological maturity, as reported for wheat (Ellis and Pieta Filho, 1992), tomato (Demir and Ellis, 1992a; Valdes and Gray, 1998; Demir and Samit, 2001; Dias et al., 2006), pepper (Demir and Ellis, 1992b) and eggplant (Demir et al., 2002). Subsequently, seed viability and vigour declines (TeKrony et al., 1980). In case of fleshy fruits, such as sweet pepper, the effect of post-harvest fruit storage on the seed quality needs to be studied. Some reports suggest that seed germination and vigour has been increased by allowing seeds to remain in harvested fruits holding seeds for sometime (Edwards and Sundstrom, 1987; Alvarenga et al., 1991; Sanchez et al., 1993; Barbedo et al., 1994; Desai et al., 1997; Vidigal et al., 2006 and Dias et al., 2006). * Author for correspondence
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During seed development and maturation, there are changes in its physical and physiological characteristics, such as size, moisture, dry matter content, germination capacity and vigour (Bewley and Black, 1994). In the orthodox seeds, final stage of the process is marked by dehydration, in such a manner that during the reserve deposition phase, there is also accumulation of potentially protective molecules, especially lea (late embryogenesis accumulated) proteins and soluble sugars, such as sucrose, raffinose, and stachyose. Their function is to prevent the damage caused by water removal from the seed tissues (Kermode, 1997; Hoekstra et al., 2001). The lea proteins are hydrophilic, stable, and are not denatured by high temperatures (Kermode, 1997). Although they are synthesized during the initial phases of seed development, when the ABA content is still high, their accumulation accelerates during dehydration, reaching the maximum at maturity (Bewley and Black, 1994). The lea proteins were first reported in developing cotton embryo (Dure and Crouch, 1981) and then, in the maturating seeds of several other species, such as soybean (Blackman et al., 1991), castor (Han et al., 1997), sugar beet (Capron et al., 2000), corn (Faria et al., 2004; Rosa et al., 2005), rice (Chourey et al., 2003) and cauliflower (Soeda et al., 2005). There are few studies regarding temporal changes in the accumulation of these proteins during seed development in fleshy vegetables. This study was done to evaluate the influence of maturation stage and post-harvest fruit storage on the accumulation of lea proteins and physiological quality of sweet pepper seeds.
Material and methods Sweet pepper plants were grown, from February to July 2007, by transplanting 40-day old seedling to a field, in Viçosa, MG, Brazil. Plant spacing was 1.2m between rows and 0.8m between plants. Before planting, fertilizer was applied according to Ribeiro et al. (1999). The plants were irrigated by sprinkler irrigation as required and were tutored using bamboo sticks. Flowers were tagged at anthesis from which layer of the plant and the fruits were harvested 40, 50, 60 or 70 days after anthesis (DAA). For each harvest time, the harvested fruits were classified according to the maturation stage based on fruits color on the outside e.g. completely green; yellow, red, and intense red, respectively. Before seed extraction, the fruits from each harvest were stored for 0, 3, 6, 9, 12 or 15 days under room conditions (about 25°C and 75% RH). The seeds were extracted manually and washed under running tap water. A sample of freshly extracted seeds was used to determine seed moisture and dry matter content by drying for 24 hours at 105 + 3°C (Brasil, 1992), and the remaining seeds were then dried under room laboratory condition to 10% moisture content (wet base). The following tests were performed: Standard germination: Four replicates of 50 seeds each were distributed over two sheets of paper towel (JProlab®) moistened with 0.2% KNO3 solution, in plastic germination boxes (11×11×4 cm) and incubated at 25°C in the dark (Anon, 1992). The normal seedlings percentage were determined 10 days (first count) and 17 days after seeding. 193
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Seedling length: Four replicates of 20 seeds each were distributed equidistantly on the upper end of the paper towel in the plastic boxes described previously. The boxes containing the seeds were placed inclined at an angle of about 45° in the germinator at 25°C. The length (mm/seedling) of the normal seedlings was measured 10 days after seeding. Accelerated aging: The seeds were uniformly distributed on a wire mesh screen suspended over 40 ml of water inside a plastic germination box (AOSA, 1983). The boxes were held in an incubator at 42 ± 0.5°C (Bhering et al., 2006), and four replicates of 50 seeds each were tested for germination as described previously. The evaluation was made 10 days after seeding and the mean normal seedling percentage was calculated. Electrical conductivity: Four replicates of 50 seeds each were weighed and then soaked in 50 ml distilled water and incubated for 24h at 25°C. The electrical conductivity of the soak solution was measured using a conductivity meter, and the results were expressed as mean μS.cm-1.g-1 seeds (ISTA, 1995). Speed emergence index (SEI): In a greenhouse, four replicates of 50 seeds each were planted 3-mm deep in a commercial planting substrate (Bioplant®) in plastic trays (230×160×160 mm). Emerged seedlings were counted daily for 30 days and SEI was calculated (Maguire, 1962). Lea protein activity: This determination was done using seeds from fresh fruits and from fruits stored for 6 or 12 days. Initially, the seeds were imbibed for five hours in a moistened paper towel at 25°C for embryo extraction. Lea proteins were extracted by grinding 100 mg of embryos in 1ml of extraction buffer (50 mM Tris-HCL-7.5; 500 mM NaCl; 5 mM MgCl2; 1mM PMSF) using a chilled mortar and pestle. The grinded triturate was transferred to 1500 μL microtubes and centrifuged (16000 G) for 30 minutes at 4°C. The supernatant was heated for 15 minutes at 85°C, in a water bath, and then centrifuged as above. The supernatant was transferred to a microtube and the pellet was discarded. For the electrophoresis, 70 μl of the embryonic axis extract was mixed with 40 μl the buffer (2.5 mL glycerol; 0.46 g SDS; 20 mg bromophenol blue) and the final volume was adjusted to 20 ml with extraction buffer. The mixture was boiled for 5 minutes and 60 μl of the mixture was applied to each well of the SDS-PAGE gel (12.5%, separator gel, 6% concentrating gel). The electrophoresis run was performed at 120 V and the gel was stained for 12 h in a solution containing 0.05% Coomassie Blue and de-stained in 10% acetic acid (Alfenas, 2006). Statistical design and analysis: The study was done in a completely randomized design and the data were analyzed as a split-plot experiment, with four replications. The harvest treatments were allocated to plots and the post-harvest storage periods to sub-plots. Data on sub-plots were subjected to regression analysis (SAS, 1989).
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Results and discussion The maximum moisture content, varying between 63.2 (fresh fruits) to 58.6% (15-day storage), was found in seeds from fruits harvested 40 DAA (figure 1). The storage of fruits harvested 50 or 70 DAA practically did not affect the seed moisture content which varied between 52.4 and 51.3% (50 DAA) and between 47.7 and 46.7% (70 DAA). However, in seeds from fresh fruits harvested 60 DAA, the moisture content of 50.2% declined to 48.9% during the initial 3-day storage, and remained unchanged during the subsequent 9 days, and then decreased to 47.7%, in the following 3 days. The moisture content of seeds that develop in the fleshy fruits, such as tomato (Demir and Ellis, 1992a; Dias et al., 2006), pepper (Demir and Ellis, 1992b), melon (Welbaum and Bradford, 1988) and eggplant (Demir et al., 2002), generally oscillates between 38 and 45%, and remains high during the entire maturation period, even after accumulation of the maximum dry matter.
Dry weight (mg/seed)
Moisture content (%)
68 64 60 56 52 48 44
(10-3) 10 8 6 4 2
0
3
6
9
12
15
Stored (days) 40 DAA: y = 62.93 – 1.17x + 0.107x2 – 0.002x3 50 DAA: y = 51.61 – 0.18x + 0.031x2 – 0.001x3 60 DAA: y = 50.20 – 0.65x + 0.095x2 – 0.004x3 70 DAA: y = 47.16 – 0.27x + 0.047x2 – 0.001x3
0
3
6
9
12
15
Stored (days) R2 = 0.876 R2 = 0.826 R2 = 0.997 R2 = 0.967
40 DAA = 4.27 + 0.024x – 0.0103x2 – 0.0007x3 R2 = 0.818 50 DAA = 6.11 + 0.16x – 0.049x2 – 0.0027x3 R2 = 0.896 60 DAA = 5.83 + 0.12x – 0.0006x2 – 0.0003x3 R2 = 0.881 70 DAA = 6.0 + 0.311x – 0.028x2 – 0.0007x3 R2 = 0.9816
Figure 1. Seed moisture content and seed dry weight for pepper fruits harvested at 40, 50, 60 and 70 DAA and stored for 0, 3, 6, 9, 12 and 15 days.
The dry matter content of seeds from fruits harvested 40 DAA remained practically constant (figure 1). The seeds from fruits harvested 50, 60 or 70 DAA had similar dry matter content. After 12 days of storage the dry matter content of seeds from fruits harvested 50 DAA declined from 0.0061 to 0.0056 mg/seed while in seeds from fruits harvested 60 or 70 DAA it increased during the first 9 days of storage, with a maximum of 0.007 mg/seed accumulating in seeds from fruits harvested 70 DAA and stored for six days. According to Carvalho and Nakagawa (2000), due to high moisture content, the seeds stored in fleshy fruits tend to respire more, consuming the reserves thus reducing dry matter content. On the other hand, there are reports of significant increase of dry matter due to fruit-to-seed nutrient transfer during storage of fleshy fruits (Alvarenga et al., 1991; Barbedo et al., 1994). Dias et al. (2006) found a small decline in seed dry matter content during post-harvest storage of tomato fruits, while Sanchez et al. (1993) reported no such differences in pepper seeds from fresh or stored fruits. 195
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The germination of seeds from early harvested fruits (40 DAA and 50 DAA) started to increase with fruit storage (figure 2), with maximum germination of seeds from fruits stored for 12 days followed by a slight decline. However, in all cases germination of seeds from fruits harvested 40 DAA remained below satisfactory levels, even after 12 days of fruit storage. Seeds extracted from fruits harvested 30 DAA (green) did not germinate despite post harvest storage of 28 days (Sanchez et al., 1993). Similar results were found for cucumber seeds obtained from early harvested fruits (20 to 35 DAA) and stored for 15 days (Barbedo et al., 1999). Germination of seeds did not improve by storing fruits harvested 60 and 70 DAA (red and intense red fruits, respectively). These results are similar to that of tomato seeds where post-harvest storage of fruits harvested 50 or 60 DAA did not affect germination (Vidigal et al., 2006), but Sanchez et al. (1993) reported germination increase of pepper seeds extracted from fruits harvested 40 DAA (green fruit) or 50 DAA (red fruit) and stored for 14 to 28 days. The data of the first count of germination showed that the seed vigour increased with fruit storage period, independent of harvest time (figure 2). The maximum germination at the first count occurred in seeds from fruits harvested 70 DAA and stored for more than 9 days. In general, the performance of seeds from fruits harvested 60 or 70 DAA was much better than those from fruits harvested 40 to 50 DAA. This was confirmed by other parameters such as seedling length, accelerated aging, SEI and electrical conductivity (figure 2). These results are similar to those reported for tomato (Vidigal et al., 2006; Dias et al., 2006), Italian pumpkin (Alvarenga et al., 1991) and watermelon (Alvarenga et al., 1984). The seedling length practically was not affected when the fruits harvested 60 DAA were stored, and in case of fruits harvested 70 DAA there was a slight increase when stored for more than 9 days (figure 2). The seedling length increased, in general, by storing fruits harvested 40 or 50 DAA, but it was less than that of seeds from fresh fruits harvested 60 or 70 DAA. The seed vigour, evaluated by the accelerated aging test, was similar for seeds from fruits harvested at 60 or 70 DAA, and was not influenced by post-harvest storage of fruits. These seeds had higher vigour than those obtained from fruits harvested at 40 or 50 DAA as also verified by seedling length (figure 2). However, the storage of fruits harvested at 50 DAA increased seed vigour, in such a way that after 15 days of storage, their vigour was similar to that of seeds from fruits harvested at 60 or 70 DAA. The trend was similar for SEI, except for the fruits harvested at 60 or 70 DAA, where SEI increased slightly after 3 to 6 days of storage and then stabilized. The decrease of electrical conductivity of the seed leachates (figure 2) from older fruits indicates improving membrane organization during the maturation process. Demir and Ellis (1992a) reported that the electrical conductivity of tomato seeds was highest at 25 DAA and lowest at 55 DAA coinciding with the maximum seed germination. In the early harvest (40 DAA), the fruit storage significantly reduced the quantity of seed leachates, because of membrane construction. This decrease was less pronounced in the subsequent harvests especially of 60 or 70 DAA, probably because the membranes were already fully structured, which suggests that in such cases post-harvest fruit storage is unnecessary. There was no change in the electrical conductivity, and consequently no improvement in 196
100
100
80
80
First count (%)
Germination (%)
SWEET PEPPER SEED MATURATION
60 40 20 0
0
3
6
9
12
60 40 20 0
15
0
3
Stored (days) 40 DAA: y = 1.76 – 7.26x + 1.88x2 – 0.08x3 R2 = 0.9637 50 DAA: y = 64.28 – 5.24x + 0.238x2 R2 = 0.9163 60 DAA: y = 82.96 – 3.15x + 0.38x2 – 0.013x3 R2 = 0.9305 70 DAA: y = 91.52 – 3.48x + 0.68x2 – 0.029x3 R2 = 0.9039
Accelerated aging (%)
Seedling length (mm)
15
60 40 20
0
3
6
9
12
15
80 60 40 20 0
0
3
Stored (days)
4 3 2 1
3
6
9
9
12
15
40 DAA: y = 0.88 – 2.09x + 0.45x2 – 0.014x3 R2 = 0.8237 50 DAA: y = –2.17 + 7.96x – 0.09x2 R2 = 0.976 60 DAA: y = 85.15 + 1.9x – 0.17x2 – 0.006x3 R2 = 0.6763 70 DAA: y = 90.32 + 1.86x – 0.43x2 – 0.019x3 R2 = 0.7674
Electrical conductivity (µS/cm/g)
5
0
6
Stored (days)
40 DAA: y = –4.95 + 6.62x + 0.279x2 R2 = 0.8261 50 DAA: y = 34.26 – 2.84x + 0.103x2 R2 = 0.9410 60 DAA: y = 59.14 – 1.11x + 0.18x2 – 0.007x3 R2 = 0.721 70 DAA: y = 53.79 – 1.87x + 0.49x2 – 0.022x3 R2 = 0.940
Speed emergency index
12
100
80
0
9
40 DAA: y = –1.35 + 0.5x + 0.075x2 R2 = 0.877 50 DAA: y = 15.67 + 4.42x – 0.077x2 R2 = 0.8915 60 DAA: y = 57.46 + 2.98x – 0.113x2 R2 = 0.7232 70 DAA: y = 45.39 + 7.03x – 0.244x2 R2 = 0.8649
100
0
6
Stored (days)
12
15
Stored (days) 40 DAA: y = –0.052 + 0.026x + 0.005x2 R2 = 0.8889 50 DAA: y = 0.6371 + 0.126x + 0.0017x2 R2 = 0.8572 60 DAA: y = 2.59 + 0.31x – 0.04x2 + 0.0019x3 R2 = 0.642 70 DAA: y = 2.35 + 0.23x – 0.033x2 + 0.0013x3 R2 = 0.742
2400 2000 1600 1200 800 400 0
3
6
9
12
15
Stored (days) 40 DAA: y = 2161.462 – 25.69x – 3.27x2 R2 = 0.8508 50 DAA: y = 1082.57 – 66.68x + 2.47x2 R2 = 0.9451 60 DAA: y = 783.58 – 37.58x + 1.49x2 R2 = 0.8706 70 DAA: y = 581.83 + 14.11x – 4.31x2 + 0.21x3 R2 = 0.948
Figure 2. Changes in germination, first count, seedling length, accelerated aging and speed emergence index and electrical conductivity of sweet pepper seeds obtained from fruits harvested at 40, 50, 60 and 70 DAA and stored for 0, 3, 6, 9, 12 and 15 days.
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seed quality. Similar results were obtained by Vidigal et al. (2006) and Dias et al. (2006) in tomato seeds obtained from fruits at different maturation stages and stored. In summary, higher vigour seeds were obtained from fruits harvested at 60 or 70 DAA, and storage of such fruits before seed extraction did not significantly improve their performance, except for the germination at first count (figure 1). On the other hand, at least 15 days of storage of fruits harvested at 50 DAA (yellow fruits) improved seed performance, to the vigour level similar to those obtained from late harvested fruits (60 or 70 DAA, red or intense red fruits, respectively). The eletrophoretic profile of proteins (figure 3) indicated absence of lea bands of about 30 kDa at the initial maturation stage (40 DAA) (black arrow), and storage of these fruits did not change the band pattern. However, lea proteins were expressed in seeds from fruits harvested at 50 DAA and stored for 12 days, although the bands were of low intensity. It coincided with the attainment of maximum vigour, as shown by the first count, accelerated aging and SEI (figure 2). In the seeds from fruits harvested 60 or 70 DAA, lea activity (50 kDa) was found during the entire storage period, with band intensity (stippled arrow) increasing from 60 DAA harvest and 6-day storage, which indicates that this protein accumulated only after this maturation stage.
30 kDa
50 kDa
M
0
6
40 DAA
12
0
6
50 DAA
12
0
6
60 DAA
12
0
6
12
70 DAA
Figure 3. Eletrophoretical profiles of lea proteins of sweet pepper seeds obtained from fruits harvested at 40, 50, 60 and 70 DAA and stored for 0, 6 and 12 days. Column M represents lea protein marker. Black and stippled arrows indicate the lea proteins molecular-weight (kDa) evaluated.
These accumulation of lea proteins coincided with the physiological alterations in the seeds indicated by germination and vigour tests (figure 2), which suggests that from 60 DAA, independent of the post-harvest fruit storage period, the seeds start to acquire tolerance to desiccation, which is associated with the attainment of physiological maturity, which generally coincides with the maximum seed quality (Bewley and Black, 1994). The electrical conductivity test (figure 2) showed that the seeds from fruits harvested at 60 DAA or later, independent of the post-harvest storage period, leaked less solutes compared to the earlier harvests, thus had adequate membrane system organization that 198
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coincided with increased lea activity (figure 3). These data strongly suggest that these proteins act as membrane protective agents as also reported by Baker et al. (1988) and Dure et al. (1989). Such proteins are highly stable, rich in glycine and other hydrophilic amino acids, and act in synergism with the soluble sugars during cytoplasm crystallization and protection of membrane surface, thus playing a structural role as protectors against the desiccation damage (Kermode, 1997). Several studies have demonstrated that the mature embryos contain high concentrations of lea proteins, which appear to be involved in acquiring tolerance to desiccation during the final stages of seed development (Galau et al., 1987; Kermode, 1997; Walters et al., 1997; Faria et al., 2004; Rosa et al., 2005). In cotton seeds the maximum lea proteins accumulated about 50 DAA, which is three days before the beginning of desiccation phase. The intensity of the activity increased in the final phase of seed maturation till they reached 25% moisture content (Galau et al., 1987). According to Silva (2006), the lea proteins in soybean seed are induced by water reduction to 60% and are directly related to the physiological seed quality. In castor bean seeds, Han et al. (1997) observed greater intensity of some lea bands at 35 days after pollinization. Faria et al. (2004) did not find lea activity in the initial developmental phases of maize seeds, which appeared and increased after the milk-3 line stage coinciding with the acquisition of tolerance to desiccation and high physiological seed quality. In synthesis, the data show that early harvest (40 DAA), even associated with 15 days of post-harvest fruit storage does not improve the physiological quality of seeds. Fruits harvested at 50 DAA must be submitted to at least 12 days of post-harvest storage to improve the physiological quality of the seeds. Such storage appears to be unnecessary for fruits harvested at 60 DAA, because the seeds have reached high physiological quality. The lea proteins were detected only after this harvest time, indicating their involvement in improving the physiological quality of pepper seeds.
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